Chimera Botulinum Toxin Type E

ABSTRACT

The present invention relates to a toxin comprising a modified light chain of a botulinum toxin type E, wherein the modified light chain comprises amino acid sequence PFVNKQFN (SEQ ID NO: 120) at the N-terminus, and amino acid sequence xDxxxLL (SEQ ID NO: 111) at the C-terminus, wherein x is any amino acid.

CROSS REFERENCE

This application is a continuation that claims priority pursuant to 35U.S.C. §120 to U.S. patent application Ser. No. 11/036,532, filed Jan.14, 2005, a continuation-in-part application which claims prioritypursuant to 35 U.S.C. §120 to U.S. patent application Ser. No.10/757,077, filed Jan. 14, 2004, a continuation-in-part of applicationwhich claims priority pursuant to 35 U.S.C. §120 to U.S. patentapplication Ser. No. 10/163,106, filed Jun. 4, 2002, acontinuation-in-part of application which claims priority pursuant to 35U.S.C. §120 to U.S. patent application Ser. No. 09/910,346, filed Jul.20, 2001; a continuation-in-part of application which claims prioritypursuant to 35 U.S.C. §120 to U.S. patent application Ser. No.09/620,840, filed Jul. 21, 2000. All prior applications are incorporatedherein by reference in their entireties.

BACKGROUND

The present invention relates to modified neurotoxins, particularlymodified Clostridial neurotoxins, and use thereof to treat variousconditions, including conditions that have been treated using naturallyoccurring botulinum toxins. For example, botulinum toxin type A has beenused in the treatment of numerous conditions including pain, skeletalmuscle conditions, smooth muscle conditions and glandular conditions.Botulinum toxins are also used for cosmetic purposes.

Numerous examples exist for treatment using botulinum toxin. Forexamples of treating pain see Aoki, et al., U.S. Pat. No. 6,113,915 andAoki, et al., U.S. Pat. No. 5,721,215. For an example of treating aneuromuscular disorder, see U.S. Pat. No. 5,053,005, which suggeststreating curvature of the juvenile spine, i.e., scoliosis, with anacetylcholine release inhibitor, preferably botulinum toxin A. For thetreatment of strabismus with botulinum toxin type A, see Elston, J. S.,et al., British Journal of Opthalmology, 1985, 69, 718-724 and 891-896.For the treatment of blepharospasm with botulinum toxin type A, seeAdenis, J. P., et al., J. Fr. Opthalmol., 1990, 13 (5) at pages 259-264.For treating spasmodic and oromandibular dystonia torticollis, seeJankovic et al., Neurology, 1987, 37, 616-623. Spasmodic dysphonia hasalso been treated with botulinum toxin type A. See Blitzer et al., Ann.Otol. Rhino. Laryngol, 1985, 94, 591-594. Lingual dystonia was treatedwith botulinum toxin type A according to Brin et al., Adv. Neurol.(1987) 50, 599-608. Cohen et al., Neurology (1987) 37 (Suppl. 1), 123-4,discloses the treatment of writers cramp with botulinum toxin type A.

It would be beneficial to have botulinum toxins with enhanced biologicalpersistence and/or enhanced biological activity.

SUMMARY

The present invention relates to a modified toxin comprising a modifiedlight chain of a botulinum toxin type E, wherein the modified lightchain comprises an amino acid sequence SEQ ID NO: 120 (PFVNKQFN) at theN-terminus, and an amino acid sequence SEQ ID NO: 112 (xExxxLL) at theC-terminus, wherein x is any amino acid.

DEFINITIONS

Before proceeding to describe the present invention, the followingdefinitions are provided and apply herein.

“Heavy chain” means the heavy chain of a Clostridial neurotoxin. It hasa molecular weight of about 100 kDa and can be referred to herein asHeavy chain or as H.

“H_(N)” means a fragment (having a molecular weight of about 50 kDa)derived from the Heavy chain of a Clostridial neurotoxin which isapproximately equivalent to the amino terminal segment of the Heavychain, or the portion corresponding to that fragment in the intact Heavychain. It is believed to contain the portion of the natural or wild-typeClostridial neurotoxin involved in the translocation of the light chainacross an intracellular endosomal membrane.

“H_(C)” means a fragment (about 50 kDa) derived from the Heavy chain ofa Clostridial neurotoxin which is approximately equivalent to thecarboxyl terminal segment of the Heavy chain, or the portioncorresponding to that fragment in the intact Heavy chain. It is believedto be immunogenic and to contain the portion of the natural or wild-typeClostridial neurotoxin involved in high affinity binding to variousneurons (including motor neurons), and other types of target cells.

“Light chain” means the light chain of a Clostridial neurotoxin. It hasa molecular weight of about 50 kDa, and can be referred to as lightchain, L or as the proteolytic domain (amino acid sequence) of aClostridial neurotoxin. The light chain is believed to be effective asan inhibitor of exocytosis, including as an inhibitor ofneurotransmitter (i.e. acetylcholine) release when the light chain ispresent in the cytoplasm of a target cell.

“Neurotoxin” means a molecule that is capable of interfering with thefunctions of a cell, including a neuron. The “neurotoxin” can benaturally occurring or man-made. The interfered with function can beexocytosis.

“Modified neurotoxin” (or “modified toxin”) means a neurotoxin whichincludes a structural modification. In other words, a “modifiedneurotoxin” is a neurotoxin which has been modified by a structuralmodification. The structural modification changes the biologicalpersistence, such as the biological half-life (i.e. the duration ofaction of the neurotoxin) and/or the biological activity of the modifiedneurotoxin relative to the neurotoxin from which the modified neurotoxinis made or derived. The modified neurotoxin is structurally differentfrom a naturally existing neurotoxin.

“Mutation” means a structural modification of a naturally occurringprotein or nucleic acid sequence. For example, in the case of nucleicacid mutations, a mutation can be a deletion, addition or substitutionof one or more nucleotides in the DNA sequence. In the case of a proteinsequence mutation, the mutation can be a deletion, addition orsubstitution of one or more amino acids in a protein sequence. Forexample, a specific amino acid comprising a protein sequence can besubstituted for another amino acid, for example, an amino acid selectedfrom a group which includes the amino acids alanine, aspargine,cysteine, aspartic acid, glutamic acid, phenylalanine, glycine,histidine, isoleucine, lysine, leucine, methionine, proline, glutamine,arginine, serine, threonine, valine, tryptophan, tyrosine or any othernatural or non-naturally occurring amino acid or chemically modifiedamino acids. Mutations to a protein sequence can be the result ofmutations to DNA sequences that when transcribed, and the resulting mRNAtranslated, produce the mutated protein sequence. Mutations to a proteinsequence can also be created by fusing a peptide sequence containing thedesired mutation to a desired protein sequence.

“Structural modification” means any change to a neurotoxin that makes itphysically or chemically different from an identical neurotoxin withoutthe structural modification.

“Biological persistence” or “persistence” means the time duration ofinterference or influence caused by a neurotoxin or a modifiedneurotoxin with a cellular (such as a neuronal) function, including thetemporal duration of an inhibition of exocytosis (such as exocytosis ofneurotransmitter, for example, acetylcholine) from a cell, such as aneuron.

“Biological half-life” or “half-life” means the time that theconcentration of a neurotoxin or a modified neurotoxin, preferably theactive portion of the neurotoxin or modified neurotoxin, for example,the light chain of Clostridial toxins, is reduced to half of theoriginal concentration in a mammalian cell, such as in a mammalianneuron.

“Biological activity” or “activity” means the amount of cellularexocytosis inhibited from a cell per unit of time, such as exocytosis ofa neurotransmitter from a neuron.

“Target cell” means a cell (including a neuron) with a binding affinityfor a neurotoxin or for a modified neurotoxin.

“PURE A” means a purified botulinum toxin type A, that is the 150 kDatoxin molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Comparison of the N-terminal sequence of LC/A (Allergan Hall A),LC/B, and LC/E. dN-LC/A shows the amino acids truncated in ourN-terminus deletion mutant. SEQ IDS NOs for the N-terminal sequences areas follows: rLC/A is SEQ ID NO: 158, dN-LC/A is SEQ ID NO: 159, LC/B isSEQ ID NO: 160, LC/E is SEQ ID NO: 161 and the consensus sequence is SEQID NO: 162.

FIG. 2: Sequence comparison of the C-terminus of the Allergan Hall ALC/A with the C-terminus of different strains of LC/E. The box containsthe di-leucine motif present on the LC/A. The sequence in that area isvery well conserved in all the LC/Es and contains two Isoleucinesinstead of the Leucines. SEQ IDS NOs for the C-terminal sequences are asfollows: BoNT/A (Hall A) is SEQ ID NO: 151, LCE (NTP genomic) is SEQ IDNO: 152, BoNT/E (Beluga) is SEQ ID NO: 153, BoNT/E (synth LC/E) is SEQID NO: 154, BoNT/E (NCTC-11219) is SEQ ID NO: 155 and the consensussequence is SEQ ID NO: 156.

FIG. 3: LC/E chimeras generated by adding the localization signals ofthe LC/A into the LC/E. Constructs were generated by site-directedmutagenesis of the di-leucine motif SEQ ID NO: 163 at the C-terminus ofthe LC/E, and by the addition of SEQ ID NO: 120 from the LC/A to theN-terminus of the LC/E. wtLC/E includes the di-leucine motif of SEQ IDNO: 163; LC/E (ExxxII) includes the di-leucine motif of SEQ ID NO: 144;LC/E (ExxxLL) includes the di-leucine motif of SEQ ID NO: 145; LC/E(NLCA) includes SEQ ID NO: 120 and di-leucine motif of SEQ ID NO: 163;LC/E (NLCA/ExxxII) includes SEQ ID NO: 120 and di-leucine motif of SEQID NO: 144; and LC/E (NLCA/ExxxLL) includes SEQ ID NO: 120 anddi-leucine motif of SEQ ID NO: 145.

FIG. 4: Catalytic activity towards the cleavage of SNAP25 of the LC/Echimeras containing the localization signals from the LC/A and expressedin SH-SY5Y cells. Two separate transfections were performed and thewestern blot data from both experiments are shown in the figure. Blotswere probed with antibody SMI-81 to the N-terminus of SNAP25.

FIG. 5: Plasmids encoding for the GFP-LC/E chimeras were transfectedinto PC-12 and SH-SY5Y cells. Three separate experiments were performed.Experiment number one is the top panel, experiment number two is themiddle panel, and experiment number three is the bottom panel. Celllysates were prepared and subjected to immunoprecipitation (top gels ineach panel) with an antibody to GFP to detect the expressed protein.Part of the lysate was used for western blots to detect catalyticactivity (bottom gels in each panel) of the chimeras expressed in cells.Each lane is numbered according to the table shown in the first panel,and reads as follow: 1. GFP negative control, 2. Wt LC/E, 3. LC/E(NLCA), 4. LC/E (ExxxII), 5. LC/E (ExxxLL), 6. LC/E (NLCA/ExxxII), 7.LC/E (NLCA/ExxxLL).

FIG. 6: Taken from PNAS publication (1). Differentiated PC-12 cellsexpressing GFP-LC/A (A) and GFP-LC/E (B).

FIG. 7: Differentiated PC-12 cells transfected with GFP-LCE(N-LCA/ExxxLL). Immunostained with antibodies to GFP, rabbit polyclonalat 1:100 (FIGS. 7A and 7C) and a combination of three Anti-SNAP₁₈₀ mousemonoclonal antibodies (1A3A7, 1G8C11 and 1C9F3), each at 1:50 dilution(FIGS. 7B and 7D). (63× magnification).

FIG. 8: Differentiated PC-12 cells transfected with GFP-LC/E(N-LCA/ExxxLL). Cells were immunostained with antibodies to GFP, mousemonoclonal at 1:100 (FIGS. 8A and 8C) and Anti-SNAP25₂₀₆ rabbitpolyclonal antibody at 1:100 (FIGS. 8B and 8D). (63× magnification).Specific cells are indicated by an arrow and are designated a, b, c, dor e. Transfected cells do not contain SNAP25₂₀₆ that is only present innon-transfected cells.

FIG. 9: Differentiated PC-12 cells transfected with native belugaGFP-LC/E, and immunostained with rabbit polyclonal antibodies to GFP(FIG. 9A) and 1:1:1 combination of anti-SNAP25₁₈₀ mouse monoclonalantibodies, each at 1:50 dilution (FIG. 9B) (63× magnification) as usedin the previous figure with the chimeric LC/E. Both images correspond tothe same cell but are not from the same plane.

FIG. 10: SH-SY5Y cells transfected with GFP-LCE construct and stainedwith anti-GFP, rabbit polyclonal at a dilution of 1:100 and secondaryanti-rabbit at 1:200. (63× magnification). A and B represent twodifferent cells from the same transfection experiment.

FIG. 11: SH-SY5Y cells transfected with GFP-LCE (N-LCA/ExxxLL) andstained with anti-GFP antibodies. (63× magnification). A and B aredifferent groups of cells from the same transfection experiment.

FIG. 12: SH-SY5Y cells transfected with GFP-LCE (N-LCA/ExxxLL). Cellswere immunostained with antibodies to anti-GFP (FIGS. 12A and 12C) anduncleaved SNAP25₂₀₆ (FIGS. 12B & 12D). (63× magnification).

FIG. 13: SH-SY5Y cells transfected with GFP-LC/E (N-LCA/ExxxLL). Cellswere immunostained with antibodies to GFP (FIG. 13A) and 1A3A7 mousemonoclonal antibody specific for SNAP25₁₈₀ (FIG. 13B). (63× mag).

FIGS. 14-a & b: Sequence of wild-type Beluga LC/E. SEQ ID NO: 136 andSEQ ID NO: 137 correspond to the amino acid sequence and the nucleicacid sequence, respectively.

FIGS. 15-a & b: Sequence of chimera LC/E with N-terminus of LC/A. SEQ IDNO: 138 and SEQ ID NO: 139 correspond to the amino acid sequence and thenucleic acid sequence, respectively.

FIGS. 16-a & b: Sequence of chimera LC/E with LC/A di-leucine motif atC-terminus. SEQ ID NO: 140 and SEQ ID NO: 141 correspond to the aminoacid sequence and the nucleic acid sequence, respectively.

FIGS. 17-a & b: Sequence of chimera LC/E with LC/A N-terminus andC-terminal di-leucine motif. SEQ ID NO: 142 and SEQ ID NO: 143correspond to the amino acid sequence and the nucleic acid sequence,respectively.

DETAILED DESCRIPTION

The present invention is based upon the discovery that the biologicalpersistence and/or the biological activity of a neurotoxin can bealtered by structurally modifying the neurotoxin. In other words, amodified neurotoxin with an altered biological persistence and/orbiological activity can be formed from a neurotoxin containing orincluding a structural modification. In some embodiments, the structuralmodification includes the fusing of a biological persistence enhancingcomponent to the primary structure of a neurotoxin to enhance itsbiological persistence. In some embodiments, the biological persistenceenhancing component is a leucine-based motif. Even more preferably, thebiological half-life and/or the biological activity of the modifiedneurotoxin is enhanced by about 100%. Generally speaking, the modifiedneurotoxin has a biological persistence of about 20% to 300% more thanan identical neurotoxin without the structural modification. That is,for example, the modified neurotoxin including the biologicalpersistence enhancing component is able to cause a substantialinhibition of neurotransmitter release for example, acetylcholine from anerve terminal for about 20% to about 300% longer than a neurotoxin thatis not modified.

The present invention also includes within its scope a modifiedneurotoxin with a biological activity altered as compared to thebiological activity of the native or unmodified neurotoxin. For example,the modified neurotoxin can exhibit a reduced or an enhanced inhibitionof exocytosis (such as exocytosis of a neurotransmitter) from a targetcell with or without any alteration in the biological persistence of themodified neurotoxin.

In a broad embodiment of the present invention, a leucine-based motif isa run of seven amino acids. The run is organized into two groups. Thefirst five amino acids starting from the amino terminal of theleucine-based motif form a “quintet of amino acids.” The two amino acidsimmediately following the quintet of amino acids form a “duplet of aminoacids.” In some embodiments, the duplet of amino acids is located at thecarboxyl terminal region of the leucine-based motif. In someembodiments, the quintet of amino acids includes at least one acidicamino acid selected from a group consisting of a glutamate and anaspartate.

The duplet of amino acid includes at least one hydrophobic amino acid,for example leucine, isoleucine, methionine, alanine, phenylalanine,tryptophan, valine or tyrosine. Preferably, the duplet of amino acid isa leucine-leucine, a leucine-isoleucine, an isoleucine-leucine or anisoleucine-isoleucine, leucine-methionine. Even more preferably, theduplet is a leucine-leucine.

In some embodiments, the leucine-based motif is xDxxxLL, (SEQ ID NO:111)wherein x can be any amino acids. In some embodiments, the leucine-basedmotif is xExxxLL, (SEQ ID NO:112) wherein E is glutamic acid. In someembodiments, the duplet of amino acids can include an isoleucine or amethionine, forming xDxxxLI (SEQ ID NO:113) or xDxxxLM, (SEQ ID NO:114)respectively. Additionally, the aspartic acid, D, can be replaced by aglutamic acid, E, to form xExxxLI, (SEQ ID NO:115) xExxxIL (SEQ IDNO:116) and xExxxLM (SEQ ID NO:117). In some embodiments, theleucine-based motif isphenylalanine-glutamate-phenylalanine-tyrosine-lysine-leucine-leucine,SEQ ID NO:118.

In some embodiments, the quintet of amino acids comprises at least onehydroxyl containing amino acid, for example, a serine, a threonine or atyrosine. Preferably, the hydroxyl containing amino acid can bephosphorylated. More preferably, the hydroxyl containing amino acid is aserine which can be phosphorylated to allow for the binding of adapterproteins.

Although non-modified amino acids are provided as examples, a modifiedamino acid is also contemplated to be within the scope of thisinvention. For example, leucine-based motif can include a halogenated,preferably, fluorinated leucine.

Various leucine-based motif are found in various species. A list ofpossible leucine-based motif derived from the various species that canbe used in accordance with this invention is shown in Table 1. This listis not intended to be limiting.

TABLE 1 Species Sequence SEQ ID NO: Botulinum type A FEFYKLL 1 Rat VMAT1 EEKRAIL 2 Rat VMAT 2 EEKMAIL 3 Rat VAChT SERDVLL 4 Rat δ VDTQVLL 5Mouse δ AEVQALL 6 Frog γ/δ SDKQNLL 7 Chicken γ/δ SDRQNLI 8 Sheep δADTQVLM 9 Human CD3γ SDKQTLL 10  Human CD4 SQIKRLL 11  Human δ ADTQALL12  S. cerevisiae Vam3p NEQSPLL 13 

VMAT is vesicular monoamine transporter; VACht is vesicularacetylcholine transporter and S. cerevisiae Vam3p is a yeast homologueof synaptobrevin. Italicized serine residues are potential sites ofphosphorylation.

The modified neurotoxin can be formed from any neurotoxin. Also, themodified neurotoxin can be formed from a fragment of a neurotoxin, forexample, a botulinum toxin with a portion of the light chain and/orheavy chain removed. Preferably, the neurotoxin used is a Clostridialneurotoxin. A Clostridial neurotoxin comprises a polypeptide havingthree amino acid sequence regions. The first amino acid sequence regioncan include a target cell (i.e. a neuron) binding moiety which issubstantially completely derived from a neurotoxin selected from a groupconsisting of baratti toxin; butyricum toxin; tetanus toxin; botulinumtype A, B, C₁, D, E, F, and G. Preferably, the first amino acid sequenceregion is derived from the carboxyl terminal region of a toxin heavychain, H_(C). Also, the first amino acid sequence region can comprise atargeting moiety which can comprise a molecule (such as an amino acidsequence) that can bind to a receptor, such as a cell surface protein orother biological component on a target cell.

The second amino acid sequence region is effective to translocate thepolypeptide or a part thereof across an endosome membrane into thecytoplasm of a neuron. In some embodiments, the second amino acidsequence region of the polypeptide comprises an amine terminal of aheavy chain, H_(N), derived from a neurotoxin selected from a groupconsisting of baratti toxin; butyricum toxin; tetanus toxin; botulinumtype A, B, C₁, D, E, F, and G.

The third amino acid sequence region has therapeutic activity when it isreleased into the cytoplasm of a target cell, such as a neuron. In someembodiments, the third amino acid sequence region of the polypeptidecomprises a toxin light chain, L, derived from a neurotoxin selectedfrom a group consisting of baratti toxin; butyricum toxin; tetanustoxin; botulinum type A, B, C₁, D, E, F, and G.

The Clostridial neurotoxin can be a hybrid neurotoxin. For example, eachof the neurotoxin's amino acid sequence regions can be derived from adifferent Clostridial neurotoxin serotype. For example, in oneembodiment, the polypeptide comprises a first amino acid sequence regionderived from the H_(C) of the tetanus toxin, a second amino acidsequence region derived from the H_(N) of botulinum type B, and a thirdamino acid sequence region derived from the light chain of botulinumserotype E. All other possible combinations are included within thescope of the present invention.

Alternatively, all three of the amino acid sequence regions of theClostridial neurotoxin can be from the same species and same serotype.If all three amino acid sequence regions of the neurotoxin are from thesame Clostridial neurotoxin species and serotype, the neurotoxin will bereferred to by the species and serotype name. For example, a neurotoxinpolypeptide can have its first, second and third amino acid sequenceregions derived from Botulinum type E. In which case, the neurotoxin isreferred as Botulinum type E.

Additionally, each of the three amino acid sequence regions can bemodified from the naturally occurring sequence from which they arederived. For example, the amino acid sequence region can have at leastone or more amino acids added or deleted as compared to the naturallyoccurring sequence.

A biological persistence enhancing component or a biological activityenhancing component, for example a leucine-based motif, can be fusedwith any of the above described neurotoxins to form a modifiedneurotoxin with an enhanced biological persistence and/or an enhancedbiological activity. “Fusing” as used in the context of this inventionincludes covalently adding to or covalently inserting in between aprimary structure of a neurotoxin. For example, a biological persistenceenhancing component and/or a biological activity enhancing component canbe added to a Clostridial neurotoxin which does not have a leucine-basedmotif in its primary structure. In some embodiments, a leucine-basedmotif is fused with a hybrid neurotoxin, wherein the third amino acidsequence is derived from botulinum serotype A, B, C₁, C₂, D, E, F, or G.In some embodiments, the leucine-based motif is fused with a botulinumtype E.

In some embodiments, a biological persistence enhancing component and/ora biological activity enhancing component is added to a neurotoxin byaltering a cloned DNA sequence encoding the neurotoxin. For example, aDNA sequence encoding a biological persistence enhancing componentand/or a biological activity enhancing component is added to a clonedDNA sequence encoding the neurotoxin into which the biologicalpersistence enhancing component and/or a biological activity enhancingcomponent is to be added. This can be done in a number of ways which arefamiliar to a molecular biologist of ordinary skill. For example, sitedirected mutagenesis or PCR cloning can be used to produce the desiredchange to the neurotoxin encoding DNA sequence. The DNA sequence canthen be reintroduced into a native host strain. In the case of botulinumtoxins the native host strain would be a Clostridium botulinum strain.Preferably, this host strain will be lacking the native botulinum toxingene. In an alternative method, the altered DNA can be introduced into aheterologous host system such as E. coli or other prokaryotes, yeast,insect cell lines or mammalian cell lines. Once the altered DNA has beenintroduced into its host, the recombinant toxin containing the addedbiological persistence enhancing component and/or a biological activityenhancing component can be produced by, for example, standardfermentation methodologies.

Similarly, a biological persistence enhancing component can be removedfrom a neurotoxin. For example, site directed mutagenesis can be used toeliminate biological persistence enhancing components, for example, aleucine-based motif.

Standard molecular biology techniques that can be used to accomplishthese and other genetic manipulations are found in Sambrook et al.(1989) which is incorporated in its entirety herein by reference.

In some embodiments, the leucine-based motif is fused with, or added to,the third amino acid sequence region of the neurotoxin. In someembodiments, the leucine-based motif is fused with, or added to, theregion towards the carboxylic terminal of the third amino acid sequenceregion. More preferably, the leucine-based motif is fused with, or addedto, the carboxylic terminal of the third region of a neurotoxin. Evenmore preferably, the leucine-based motif is fused with, or added to thecarboxylic terminal of the third region of botulinum type E. The thirdamino acid sequence to which the leucine-based motif is fused or addedcan be a component of a hybrid or chimeric modified neurotoxin. Forexample, the leucine-based motif can be fused to or added to the thirdamino acid sequence region (or a part thereof) of one botulinum toxintype (i.e. a botulinum toxin type A), where the leucine-basedmotif-third amino acid sequence region has itself been fused to orconjugated to first and second amino acid sequence regions from anothertype (or types) of a botulinum toxin (such as botulinum toxin type Band/or E).

In some embodiments, a structural modification of a neurotoxin which hasa pre-existing biological persistence enhancing component and/or abiological activity enhancing component, for example, a leucine-basedmotif includes deleting or substituting one or more amino acids of theleucine-based motif. In addition, a modified neurotoxin includes astructural modification which results in a neurotoxin with one or moreamino acids deleted or substituted in the leucine-based motif. Theremoval or substitution of one or more amino acids from the preexistingleucine-based motif is effective to reduce the biological persistenceand/or a biological activity of a modified neurotoxin. For example, thedeletion or substitution of one or more amino acids of the leucine-basedmotif of botulinum type A reduces the biological half-life and/or thebiological activity of the modified neurotoxin.

Amino acids that can be substituted for amino acids contained in abiological persistence enhancing component include alanine, aspargine,cysteine, aspartic acid, glutamic acid, phenylalanine, glycine,histidine, isoleucine, lysine, leucine, methionine, proline, glutamine,arginine, serine, threonine, valine, tryptophan, tyrosine and othernaturally occurring amino acids as well as non-standard amino acids.

In the present invention the native botulinum type A light chain hasbeen shown to localize to differentiated PC12 cell membranes in acharacteristic pattern. Biological persistence enhancing components areshown to substantially contribute to this localization.

The data of the present invention demonstrates that when the botulinumtoxin type A light chain is truncated or when the leucine-based motif ismutated, the light chain substantially loses its ability to localize tothe membrane in its characteristic pattern. Localization to the cellularmembrane is believed to be a key factor in determining the biologicalpersistence and/or the biological activity of a botulinum toxin. This isbecause localization to a cell membrane can protect the localizedprotein from intracellular protein degradation.

The deletion of the leucine-based motif from the light chain ofbotulinum type A can change membrane localization of the type A lightchain. GFP fusion proteins were produced and visualized indifferentiated PC12 cells using methods well known to those skilled inthe art, for example, as described in Galli et al (1998) Mol Biol Cell9:1437-1448, incorporated in its entirety herein by reference; also, forexample, as described in Martinez-Arca et al (2000) J Cell Biol149:889-899, also incorporated in its entirety herein by reference.

Further studies have been done in the present invention to analyze theeffect of specific amino acid substitutions within the leucine-basedmotif. For example, in one study both leucine residues contained in theleucine-based motif were substituted for alanine residues. Thesubstitution of alanine for leucine at positions 427 and 428 in thebotulinum type A light chain substantially changes the localizationcharacteristic of the light chain.

It is within the scope of this invention that a leucine-based motif, orany other persistence enhancing component and/or a biological activityenhancing component present on a light chain, can be used to protect theheavy chain as well. A random coil belt extends from the botulinum typeA translocation domain and encircles the light chain. It is possiblethat this belt keeps the two subunits in proximity to each other insidethe cell while the light chain is localized to the cell membrane.

In addition, the data of the present invention shows that theleucine-based motif can be valuable in localizing the botulinum A toxinin close proximity to the SNAP-25 substrate within the cell. This canmean that the leucine-based motif is important not only for determiningthe half-life of the toxin but for determining the activity of the toxinas well. That is, the toxin will have a greater activity if it ismaintained in close proximity to the SNAP-25 substrate inside the cell.Dong et al., PNAS, 101(41): 14701-14706, 2004.

The data of the present invention clearly shows that truncation of thelight chain, thereby deleting the leucine-based motif, or amino acidsubstitution within the leucine-based motif substantially changesmembrane localization of the botulinum type A light chain in nervecells. In both truncation and substitution a percentage of the alteredlight chain can localize to the cell membrane in a pattern unlike thatof the native type A light chain. This data supports the presence ofbiological persistence enhancing components other than a leucine-basedmotif such as tyrosine motifs and amino acid derivatives. Use of theseother biological persistence enhancing components and/or a biologicalactivity enhancing components in modified neurotoxins is also within thescope of the present invention.

Also within the scope of the present invention is more than onebiological persistence enhancing component used in combination in amodified neurotoxin to alter biological persistence of the neurotoxinthat is modified. The present invention also includes use of more thanone biological activity enhancing or biological activity reducingcomponents used in combination in a modified neurotoxin to alter thebiological activity of the neurotoxin that is modified.

Tyrosine-based motifs are within the scope of the present invention asbiological persistence and/or a biological activity altering components.Tyrosine-based motifs comprise the sequence Y-X-X-Hy (SEQ ID NO:119)where Y is tyrosine, X is any amino acid and Hy is a hydrophobic aminoacid. Tyrosine-based motifs can act in a manner that is similar to thatof leucine-based motifs.

Also within the scope of the present invention are modified neurotoxinswhich comprise one or more biological persistence altering componentsand/or a biological activity enhancing components which occur naturallyin both botulinum toxin types A and B.

Amino acid derivatives are also within the scope of the presentinvention as biological persistence enhancing components and/or asbiological activity enhancing components. Examples of amino acidderivatives that act to effect biological persistence and/or biologicalactivity are phosphorylated amino acids. These amino acids include, forexample, amino acids phosphorylated by tyrosine kinase, protein kinase Cor casein kinase II. Other amino acid derivatives within the scope ofthe present invention as biological persistence enhancing componentsand/or as biological activity enhancing components are myristylatedamino acids and N-glycosylated amino acids.

The present invention also contemplates compositions which include abotulinum light chain component interacting with a cellular structurecomponent, for example, an intracellular structure component. Thestructure component may include lipid, carbohydrate, protein or nucleicacid or any combination thereof.

The structure component may include a cell membrane, for example, aplasma membrane. In certain embodiments, the structure componentcomprises all or part of one or more organelles, for example, thenucleus, endoplasmic reticulum, golgi apparatus, mitochondria, lysosomesor secretory vesicles or combinations thereof. The structure componentmay include any portion of an organelle, for example, the membrane of anorganelle. The structure component may also include any substance whichis included in the cytoplasm of a cell.

The structure component may include one or more proteins. In someembodiments, the structure component includes one or more cellularproteins. One or more of these cellular proteins may be membraneassociated proteins, for example, plasma membrane associated proteins.In some embodiments of the invention, the structure component includesadaptor proteins. Examples of adaptor proteins are AP-1, AP-2 and AP-3.Adaptor proteins and their characteristics are well known in the art andare discussed in, for example, Darsow et al., J. Cell Bio., 142, 913(1998) which is incorporated in its entirety herein by reference. Theone or more proteins may also include the substrate which is cleaved bythe proteolytic domain of a botulinum toxin light chain component. Forexample, a protein included in the structure component may be SNAP-25.

The interaction between the light chain of botulinum type A and thestructure component may contribute to localization of the toxin in acertain pattern. Therefore, the interaction may act to facilitateproteolysis by, for example, increasing the biological persistenceand/or biological activity of the light chain.

A botulinum toxin heavy chain or portion thereof may also be associatedwith the light chain component when the light chain is interacting withthe structure component.

In some embodiments, a botulinum toxin light chain component, wheninteracting with the structure component in a cell, may localize in thecell in a particular pattern. For example, localization of a botulinumtoxin type A light chain component may be in a unctuate or spottedpattern. For example, a botulinum type A light chain component may belocalized in a unctuate pattern on a cell membrane, for example, aplasma membrane. Botulinum type B light chain may localize in thecytoplasm. Botulinum type E may localize to the plasma membrane but to alesser degree than type A. Botulinum type E may also localize in thecytoplasm.

Methodologies to produce an isolated composition of the invention areavailable to those skilled in the art. For example, a composition may beisolated by isolating the plasma membrane from a cell after introductionof a light chain component, for example, light chain A, into a cell. Thelight chain may be introduced into the cell by, for example,electroporation or by endocytosis. In the case of introduction into thecell by endocytosis, a heavy chain component may be included with thelight chain component to facilitate the endocytosis, for example,receptor mediated endocytosis, of the light chain. In such preparationprocess, the heavy chain component may also be isolated and be includedin the composition.

After introduction into the cell, the light chain component associatesor interacts with the substrate component forming a composition. Thecomposition may be isolated by purification of the light chaincomponent-structure component from the cell. Standard purificationtechniques known to those skilled in the art may be used to isolate amembrane and/or membrane associated protein(s) which is included in thestructure component which interacts with the light chain component.Examples of conventional techniques for isolation and purification ofthe light chain component/structure component includeimmunoprecipitation and/or membrane purification techniques.

The light chain component may be crosslinked to a portion of thestructure component before isolation. The technical procedures for crosslinking of biomolecules using agents such as DTBP are well known tothose skilled in the art.

In some embodiments, a composition of the invention may be prepared bymixing together a purified or a partially purified light chain componentand a purified or a partially purified intracellular structure componentunder conditions which are effective to form the composition. Conditionsimportant in forming the composition may include Ph, ionic concentrationand temperature.

The botulinum toxin light chain component of a composition, may be amodified botulinum toxin light chain. Modifications may be mutationsand/or deletions as described elsewhere herein.

A modified light chain component may include a light chain A modified toremove a leucine based motif or other structure(s) which contributes tolocalization of the type A light chain to the plasma membrane therebyresulting in a light chain with a reduced ability to localize to aplasma membrane. This may result in a reduction in the biologicalactivity and/or biological persistence of the light chain A. Thebiological persistence and/or activity of the modified light chain maybe about 10% to about 90% that of an unmodified type A light chain.

Another modified light chain component may include a light chain Amodified by adding one or more leucine based motifs, or otherstructure(s) which contributes to localization of the type A light chainto the plasma membrane, thereby resulting in a light chain with anincreased ability to localize to a plasma membrane. This may result inan increase in the biological activity and/or biological persistence ofthe light chain A. The biological persistence and/or activity of themodified light chain may be about 1.5 to about 5 times that of anunmodified type A light chain.

A modified light chain component may include a light chain E modified byadding one or more leucine based motifs, or other structure(s) whichcontribute to localization of the type A light chain to the plasmamembrane, thereby resulting in a light chain with an increased abilityto localize to a plasma membrane. This may result in an increase in thebiological activity and/or biological persistence of the light chain E.The biological persistence and/or activity of the modified light chainmay be about 2 to about 20 times that of an unmodified type E lightchain.

Compositions of the invention have many uses and applications, forexample, in research science and medicine. Other uses and applicationswill be readily apparent to those skilled in the art.

In one broad aspect of the present invention, a method is provided fortreating a condition using a modified neurotoxin. The conditions caninclude, for example, skeletal muscle conditions, smooth muscleconditions, pain and glandular conditions. The modified neurotoxin canalso be used for cosmetics, for example, to treat brow furrows.

The neuromuscular disorders and conditions that can be treated with amodified neurotoxin include: for example, spasmodic dysphonia, laryngealdystonia, oromandibular and lingual dystonia, cervical dystonia, focalhand dystonia, blepharospasm, strabismus, hemifacial spasm, eyeliddisorders, spasmodic torticolis, cerebral palsy, focal spasticity andother voice disorders, spasmodic colitis, neurogenic bladder, anismus,limb spasticity, tics, tremors, bruxism, anal fissure, achalasia,dysphagia and other muscle tone disorders and other disorderscharacterized by involuntary movements of muscle groups can be treatedusing the present methods of administration. Other examples ofconditions that can be treated using the present methods andcompositions are lacrimation, hyperhydrosis, excessive salivation andexcessive gastrointestinal secretions, as well as other secretorydisorders. In addition, the present invention can be used to treatdermatological conditions, for example, reduction of brow furrows,reduction of skin wrinkles. The present invention can also be used inthe treatment of sports injuries.

Borodic U.S. Pat. No. 5,053,005 discloses methods for treating juvenilespinal curvature, i.e. scoliosis, using botulinum type A. The disclosureof Borodic is incorporated in its entirety herein by reference. In someembodiments, using substantially similar methods as disclosed byBorodic, a modified neurotoxin can be administered to a mammal,preferably a human, to treat spinal curvature. In some embodiments, amodified neurotoxin comprising botulinum type E fused with aleucine-based motif is administered. Even more preferably, a modifiedneurotoxin comprising botulinum type A-E with a leucine-based motiffused to the carboxyl terminal of its light chain is administered to themammal, preferably a human, to treat spinal curvature.

In addition, the modified neurotoxin can be administered to treat otherneuromuscular disorders using well known techniques that are commonlyperformed with botulinum type A. For example, the present invention canbe used to treat pain, for example, headache pain, pain from musclespasms and various forms of inflammatory pain. For example, Aoki U.S.Pat. No. 5,721,215 and Aoki U.S. Pat. No. 6,113,915 disclose methods ofusing botulinum toxin type A for treating pain. The disclosure of thesetwo patents is incorporated in its entirety herein by reference.

Autonomic nervous system disorders can also be treated with a modifiedneurotoxin. For example, glandular malfunctioning is an autonomicnervous system disorder. Glandular malfunctioning includes excessivesweating and excessive salivation. Respiratory malfunctioning is anotherexample of an autonomic nervous system disorder. Respiratorymalfunctioning includes chronic obstructive pulmonary disease andasthma. Sanders et al. disclose methods for treating the autonomicnervous system; for example, treating autonomic nervous system disorderssuch as excessive sweating, excessive salivation, asthma, etc., usingnaturally existing botulinum toxins. The disclosure of Sander et al. isincorporated in its entirety by reference herein. In some embodiments,substantially similar methods to that of Sanders et al. can be employed,but using a modified neurotoxin, to treat autonomic nervous systemdisorders such as the ones discussed above. For example, a modifiedneurotoxin can be locally applied to the nasal cavity of the mammal inan amount sufficient to degenerate cholinergic neurons of the autonomicnervous system that control the mucous secretion in the nasal cavity.

Pain that can be treated by a modified neurotoxin includes pain causedby muscle tension, or spasm, or pain that is not associated with musclespasm. For example, Binder in U.S. Pat. No. 5,714,468 discloses thatheadache caused by vascular disturbances, muscular tension, neuralgiaand neuropathy can be treated with a naturally occurring botulinumtoxin, for example Botulinum type A. The disclosures of Binder areincorporated in its entirety herein by reference. In some embodiments,substantially similar methods to that of Binder can be employed, butusing a modified neurotoxin, to treat headache, especially the onescaused by vascular disturbances, muscular tension, neuralgia andneuropathy. Pain caused by muscle spasm can also be treated by anadministration of a modified neurotoxin. For example, a botulinum type Efused with a leucine-based motif, preferably at the carboxyl terminal ofthe botulinum type E light chain, can be administered intramuscularly atthe pain/spasm location to alleviate pain.

Furthermore, a modified neurotoxin can be administered to a mammal totreat pain that is not associated with a muscular disorder, such asspasm. In one broad embodiment, methods of the present invention totreat non-spasm related pain include central administration orperipheral administration of the modified neurotoxin.

For example, Foster et al. in U.S. Pat. No. 5,989,545 discloses that abotulinum toxin conjugated with a targeting moiety can be administeredcentrally (intrathecally) to alleviate pain. The disclosures of Fosteret al. are incorporated in its entirety by reference herein. In someembodiments, substantially similar methods to that of Foster et al. canbe employed, but using the modified neurotoxin according to thisinvention, to treat pain. The pain to be treated can be an acute pain,or preferably, chronic pain.

An acute or chronic pain that is not associated with a muscle spasm canalso be alleviated with a local, peripheral administration of themodified neurotoxin to an actual or a perceived pain location on themammal. In some embodiments, the modified neurotoxin is administeredsubcutaneously at or near the location of pain, for example, at or neara cut. In some embodiments, the modified neurotoxin is administeredintramuscularly at or near the location of pain, for example, at or neara bruise location on the mammal. In some embodiments, the modifiedneurotoxin is injected directly into a joint of a mammal, for treatingor alleviating pain caused by arthritic conditions. Also, frequentrepeated injection or infusion of the modified neurotoxin to aperipheral pain location is within the scope of the present invention.However, given the long lasting therapeutic effects of the presentinvention, frequent injection or infusion of the neurotoxin can not benecessary. For example, practice of the present invention can provide ananalgesic effect, per injection, for 2 months or longer, for example 27months, in humans.

Without wishing to limit the invention to any mechanism or theory ofoperation, it is believed that when the modified neurotoxin isadministered locally to a peripheral location, it inhibits the releaseof Neuro-substances, for example substance P, from the peripheralprimary sensory terminal by inhibiting SNARE-dependent exocytosis. Sincethe release of substance P by the peripheral primary sensory terminalcan cause or at least amplify pain transmission process, inhibition ofits release at the peripheral primary sensory terminal will dampen thetransmission of pain signals from reaching the brain.

The amount of the modified neurotoxin administered can vary widelyaccording to the particular disorder being treated, its severity andother various patient variables including size, weight, age, andresponsiveness to therapy. Generally, the dose of modified neurotoxin tobe administered will vary with the age, presenting condition and weightof the mammal, preferably a human, to be treated. The potency of themodified neurotoxin will also be considered.

Assuming a potency (for a botulinum toxin type A) which is substantiallyequivalent to LD₅₀=2,730 U in a human patient and an average person is75 kg, a lethal dose (for a botulinum toxin type A) would be about 36U/kg of a modified neurotoxin. Therefore, when a modified neurotoxinwith such an LD₅₀ is administered, it would be appropriate to administerless than 36 U/kg of the modified neurotoxin into human subjects.Preferably, about 0.01 U/kg to 30 U/kg of the modified neurotoxin isadministered. More preferably, about 1 U/kg to about 15 U/kg of themodified neurotoxin is administered. Even more preferably, about 5 U/kgto about 10 U/kg modified neurotoxin is administered. Generally, themodified neurotoxin will be administered as a composition at a dosagethat is proportionally equivalent to about 2.5 cc/100 U. Those ofordinary skill in the art will know, or can readily ascertain, how toadjust these dosages for neurotoxin of greater or lesser potency. It isknown that botulinum toxin type B can be administered at a level aboutfifty times higher that that used for a botulinum toxin type A forsimilar therapeutic effect. Thus, the units amounts set forth above canbe multiplied by a factor of about fifty for a botulinum toxin type B.

Although examples of routes of administration and dosages are provided,the appropriate route of administration and dosage are generallydetermined on a case by case basis by the attending physician. Suchdeterminations are routine to one of ordinary skill in the art (see forexample, Harrison's Principles of Internal Medicine (1998), edited byAnthony Fauci et al., 14^(th) edition, published by McGraw Hill). Forexample, the route and dosage for administration of a modifiedneurotoxin according to the present disclosed invention can be selectedbased upon criteria such as the solubility characteristics of themodified neurotoxin chosen as well as the types of disorder beingtreated.

The modified neurotoxin can be produced by chemically linking theleucine-based motif to a neurotoxin using conventional chemical methodswell known in the art. For example, botulinum type E can be obtained byestablishing and growing cultures of Clostridium botulinum in afermenter, and then harvesting and purifying the fermented mixture inaccordance with known procedures.

The modified neurotoxin can also be produced by recombinant techniques.Recombinant techniques are preferable for producing a neurotoxin havingamino acid sequence regions from different Clostridial species or havingmodified amino acid sequence regions. Also, the recombinant technique ispreferable in producing botulinum type A with the leucine-based motifbeing modified by deletion. The technique includes steps of obtaininggenetic materials from natural sources, or synthetic sources, which havecodes for a cellular binding moiety, an amino acid sequence effective totranslocate the neurotoxin or a part thereof, and an amino acid sequencehaving therapeutic activity when released into a cytoplasm of a targetcell, preferably a neuron. In some embodiments, the genetic materialshave codes for the biological persistence enhancing component,preferably the leucine-based motif, the H_(C), the H_(N) and the lightchain of the Clostridial neurotoxins and fragments thereof. The geneticconstructs are incorporated into host cells for amplification by firstfusing the genetic constructs with a cloning vectors, such as phages orplasmids. Then the cloning vectors are inserted into a host, forexample, Clostridium sp., E. coli or other prokaryotes, yeast, insectcell line or mammalian cell lines. Following the expressions of therecombinant genes in host cells, the resultant proteins can be isolatedusing conventional techniques.

There are many advantages to producing these modified neurotoxinsrecombinantly. For example, to form a modified neurotoxin, a modifyingfragment, or component must be attached or inserted into a neurotoxin.The production of neurotoxin from anaerobic Clostridium cultures is acumbersome and time-consuming process including a multi-steppurification protocol involving several protein precipitation steps andeither prolonged and repeated crystallization of the toxin or severalstages of column chromatography. Significantly, the high toxicity of theproduct dictates that the procedure must be performed under strictcontainment (BL-3). During the fermentation process, the foldedsingle-chain neurotoxins are activated by endogenous Clostridialproteases through a process termed nicking to create a dichain.Sometimes, the process of nicking involves the removal of approximately10 amino acid residues from the single-chain to create the dichain formin which the two chains remain covalently linked through the intrachaindisulfide bond.

The nicked neurotoxin is much more active than the unnicked form. Theamount and precise location of nicking varies with the serotypes of thebacteria producing the toxin. The differences in single-chain neurotoxinactivation and, hence, the yield of nicked toxin, are due to variationsin the serotype and amounts of proteolytic activity produced by a givenstrain. For example, greater than 99% of Clostridial botulinum serotypeA single-chain neurotoxin is activated by the Hall A Clostridialbotulinum strain, whereas serotype B and E strains produce toxins withlower amounts of activation (0 to 75% depending upon the fermentationtime). Thus, the high toxicity of the mature neurotoxin plays a majorpart in the commercial manufacture of neurotoxins as therapeutic agents.

The degree of activation of engineered Clostridial toxins is, therefore,an important consideration for manufacture of these materials. It wouldbe a major advantage if neurotoxins such as botulinum toxin and tetanustoxin could be expressed, recombinantly, in high yield inrapidly-growing bacteria (such as heterologous E. coli cells) asrelatively non-toxic single-chains (or single chains having reducedtoxic activity) which are safe, easy to isolate and simple to convert tothe fully-active form.

With safety being a prime concern, previous work has concentrated on theexpression in E. coli and purification of individual H and light chainsof tetanus and botulinum toxins; these isolated chains are, bythemselves, non-toxic; see Li et al., Biochemistry 33:7014-7020 (1994);Zhou et al., Biochemistry 34:15175-15181 (1995), hereby incorporated byreference herein. Following the separate production of these peptidechains and under strictly controlled conditions the H and light chainscan be combined by oxidative disulphide linkage to form theneuroparalytic di-chains.

EXAMPLES

The following non-limiting examples provide those of ordinary skill inthe art with specific suitable methods to treat non-spasm related painwithin the scope of the present invention and are not intended to limitthe scope of the invention.

Example 1 Treatment of Pain Associated with Muscle Disorder

An unfortunate 36 year old woman has a 15 year history oftemporomandibular joint disease and chronic pain along the masseter andtemporalis muscles. Fifteen years prior to evaluation she notedincreased immobility of the jaw associated with pain and jaw opening andclosing and tenderness along each side of her face. The left side isoriginally thought to be worse than the right. She is diagnosed ashaving temporomandibular joint (TMJ) dysfunction with subluxation of thejoint and is treated with surgical orthoplasty meniscusectomy andcondyle resection.

She continues to have difficulty with opening and closing her jaw afterthe surgical procedures and for this reason, several years later, asurgical procedure to replace prosthetic joints on both sides isperformed. After the surgical procedure progressive spasms and deviationof the jaw ensues. Further surgical revision is performed subsequent tothe original operation to correct prosthetic joint loosening. The jawcontinues to exhibit considerable pain and immobility after thesesurgical procedures. The TMJ remained tender as well as the muscleitself. There are tender points over the temporomandibular joint as wellas increased tone in the entire muscle. She is diagnosed as havingpost-surgical myofascial pain syndrome and is injected with the modifiedneurotoxin into the masseter and temporalis muscles; the modifiedneurotoxin is botulinum type E comprising a leucine-based motif. Theparticular dose as well as the frequency of administrations depends upona variety of factors within the skill of the treating physician.

Several days after the injections she noted substantial improvement inher pain and reports that her jaw feels looser. This gradually improvesover a 2 to 3 week period in which she notes increased ability to openthe jaw and diminishing pain. The patient states that the pain is betterthan at any time in the last 4 years. The improved condition persistsfor up to 27 months after the original injection of the modifiedneurotoxin.

Example 2 Treatment of Pain Subsequent to Spinal Cord Injury

A patient, age 39, experiencing pain subsequent to spinal cord injury istreated by intrathecal administration, for example, by spinal tap or bycatherization (for infusion) to the spinal cord, with the modifiedneurotoxin; the modified neurotoxin is botulinum type E comprising aleucine-based motif. The particular toxin dose and site of injection, aswell as the frequency of toxin administrations, depend upon a variety offactors within the skill of the treating physician, as previously setforth. Within about 1 to about 7 days after the modified neurotoxinadministration, the patient's pain is substantially reduced. The painalleviation persists for up to 27 months.

Example 3 Peripheral Administration of a Modified Neurotoxin to Treat“Shoulder-Hand Syndrome”

Pain in the shoulder, arm, and hand can develop, with musculardystrophy, osteoporosis and fixation of joints. While most common aftercoronary insufficiency, this syndrome can occur with cervicalosteoarthritis or localized shoulder disease, or after any prolongedillness that requires the patient to remain in bed.

A 46 year old woman presents a shoulder-hand syndrome type pain. Thepain is particularly localized at the deltoid region. The patient istreated by a bolus injection of a modified neurotoxin subcutaneously tothe shoulder; preferably the modified neurotoxin is botulinum type Ecomprising a leucine-based motif. The modified neurotoxin can also be,for example, modified botulinum type A, B, C1, C2, D, E, F or G whichcomprise a leucine-based motif. The particular dose as well as thefrequency of administrations depends upon a variety of factors withinthe skill of the treating physician, as previously set forth. Within 1-7days after modified neurotoxin administration the patient's pain issubstantially alleviated. The duration of the pain alleviation is fromabout 7 to about 27 months.

Example 4 Peripheral Administration of a Modified Neurotoxin to TreatPostherapeutic Neuralgia

Postherapeutic neuralgia is one of the most intractable of chronic painproblems. Patients suffering this excruciatingly painful process oftenare elderly, have debilitating disease, and are not suitable for majorinterventional procedures. The diagnosis is readily made by theappearance of the healed lesions of herpes and by the patient's history.The pain is intense and emotionally distressing. Postherapeuticneuralgia can occur anywhere, but is most often in the thorax.

A 76 year old man presents a postherapeutic type pain. The pain islocalized to the abdomen region. The patient is treated by a bolusinjection of a modified neurotoxin intradermally to the abdomen; themodified neurotoxin is, for example, botulinum type A, B, C1, C2, D, E,F and/or G. The modified neurotoxin comprises a leucine-based motifand/or additional tyrosine-based motifs. The particular dose as well asthe frequency of administration depends upon a variety of factors withinthe skill of the treating physician, as previously set forth. Within 1-7days after modified neurotoxin administration the patient's pain issubstantially alleviated. The duration of the pain alleviation is fromabout 7 to about 27 months.

Example 5 Peripheral Administration of a Modified Neurotoxin to TreatNasopharyngeal Tumor Pain

These tumors, most often squamous cell carcinomas, are usually in thefossa of Rosenmuller and can invade the base of the skull. Pain in theface is common. It is constant, dull-aching in nature.

A 35 year old man presents a nasopharyngeal tumor type pain. Pain isfound at the lower left cheek. The patient is treated by a bolusinjection of a modified neurotoxin intramuscularly to the cheek,preferably the modified neurotoxin is botulinum type A, B, C1, C2, D, E,F or G comprising additional biological persistence enhancing amino acidderivatives, for example, tyrosine phosphorylations. The particular doseas well as the frequency of administrations depends upon a variety offactors within the skill of the treating physician. Within 1-7 daysafter modified neurotoxin administration the patient's pain issubstantially alleviated. The duration of the pain alleviation is fromabout 7 to about 27 months.

Example 6 Peripheral Administration of a Modified Neurotoxin to TreatInflammatory Pain

A patient, age 45, presents an inflammatory pain in the chest region.The patient is treated by a bolus injection of a modified neurotoxinintramuscularly to the chest, preferably the modified neurotoxin isbotulinum type A, B, C1, C2, D, E, F or G comprising additionaltyrosine-based motifs. The particular dose as well as the frequency ofadministrations depends upon a variety of factors within the skill ofthe treating physician, as previously set forth. Within 1-7 days aftermodified neurotoxin administration the patient's pain is substantiallyalleviated. The duration of the pain alleviation is from about 7 toabout 27 months.

Example 7 Treatment of Excessive Sweating

A male, age 65, with excessive unilateral sweating is treated byadministering a modified neurotoxin. The dose and frequency ofadministration depends upon degree of desired effect. Preferably, themodified neurotoxin is botulinum type A, B, C1, C2, D, E, F and/or G.The modified neurotoxins comprise a leucine-based motif. Theadministration is to the gland nerve plexus, ganglion, spinal cord orcentral nervous system. The specific site of administration is to bedetermined by the physician's knowledge of the anatomy and physiology ofthe target glands and secretory cells. In addition, the appropriatespinal cord level or brain area can be injected with the toxin. Thecessation of excessive sweating after the modified neurotoxin treatmentis up to 27 months.

Example 8 Post Surgical Treatments

A female, age 22, presents a torn shoulder tendon and undergoesorthopedic surgery to repair the tendon. After the surgery, the patientis administered intramuscularly with a modified neurotoxin to theshoulder. The modified neurotoxin can botulinum type A, B, C, D, E, F,and/or G wherein one or more amino acids of a biological persistenceenhancing component are deleted from the toxin. For example, one or moreleucine residues can be deleted from and/or mutated from theleucine-based motif in botulinum toxin serotype A. Alternatively, one ormore amino acids of the leucine-based motif can be substituted for otheramino acids. For example, the two leucines in the leucine-based motifcan be substituted for alanines. The particular dose as well as thefrequency of administrations depends upon a variety of factors withinthe skill of the treating physician. The specific site of administrationis to be determined by the physician's knowledge of the anatomy andphysiology of the muscles. The administered modified neurotoxin reducesmovement of the arm to facilitate the recovery from the surgery. Theeffect of the modified neurotoxin is for about five weeks or less.

Example 9 Cloning, Expression and Purification of the BotulinumNeurotoxin Light Chain Gene

This example describes methods to clone and express a DNA nucleotidesequence encoding a botulinum toxin light chain and purify the resultingprotein product. A DNA sequence encoding the botulinum toxin light chaincan be amplified by PCR protocols which employ syntheticoligonucleotides having sequences corresponding to the 5′ and 3′ endregions of the light chain gene. Design of the primers can allow for theintroduction of restriction sites, for example, Stu I and EcoR Irestriction sites into the 5′ and 3′ ends of the botulinum toxin lightchain gene PCR product. These restriction sites can be subsequently usedto facilitate unidirectional subcloning of the amplification products.Additionally, these primers can introduce a stop codon at the C-terminusof the light chain coding sequence. Chromosomal DNA from C. botulinum,for example, strain HaIIA, can serve as a template in the amplificationreaction.

The PCR amplification can be performed in a 0.1 mL volume containing 10mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 0.2 mM of eachdeoxynucleotide triphosphate (dNTP), 50 μmol of each primer, 200 ng ofgenomic DNA and 2.5 units of Taq DNA polymerase. The reaction mixturecan be subjected to 35 cycles of denaturation (1 minute at 94° C.),annealing (2 minutes at 55° C.) and polymerization (2 minutes at 72°C.). Finally, the reaction can be extended for an additional 5 minutesat 72° C.

The PCR amplification product can be digested with for example, Stu Iand EcoR I, to release the light chain encoding, cloned, PCR DNAfragment. This fragment can then be purified by, for example, agarosegel electrophoresis, and ligated into, for example, a Sma I and EcoR Idigested pBluescript II SK phagemid. Bacterial transformants, forexample, E. coli, harboring this recombinant phagemid can be identifiedby standard procedures, such as blue/white screening. Clones comprisingthe light chain encoding DNA can be identified by DNA sequence analysisperformed by standard methods. The cloned sequences can be confirmed bycomparing the cloned sequences to published sequences for botulinumlight chains, for example, Binz, et al., in J. Biol. Chem. 265, 9153(1990), Thompson et al., in Eur. J. Biochem. 189, 73 (1990) and Minton,Clostridial Neurotoxins, The Molecular Pathogenesis of Tetanus andBotulism p. 161-191, Edited by C. Motecucco (1995).

The light chain can be subcloned into an expression vector, for example,pMaI-P2. pMaI-P2 harbors the maIE gene encoding MBP (maltose bindingprotein) which is controlled by a strongly inducible promoter, P_(tac).

To verify expression of the botulinum toxin light chain, a well isolatedbacterial colony harboring the light chain gene containing pMaI-P2 canbe used to inoculate L-broth containing 0.1 mg/ml ampicillin and 2%(w/v) glucose, and grown overnight with shaking at 30° C. The overnightcultures can be diluted 1:10 into fresh L-broth containing 0.1 mg/ml ofampicillin and incubated for 2 hours. Fusion protein expression can beinduced by addition of IPTG to a final concentration of 0.1 mM. After anadditional 4 hour incubation at 30° C., bacteria can be collected bycentrifugation at 6,000×g for 10 minutes.

A small-scale SDS-PAGE analysis can confirm the presence of a 90 kDaprotein band in samples derived from IPTG-induced bacteria. This MWwould be consistent with the predicted size of a fusion protein havingMBP (˜40 kDa) and botulinum toxin light chain (˜50 kDa) components.

The presence of the desired fusion proteins in IPTG-induced bacterialextracts can be confirmed by western blotting using the polyclonalanti-L chain probe described by Cenci di Bello et al., in Eur. J.Biochem. 219, 161 (1993). Reactive bands on PVDF membranes (Pharmacia;Milton Keynes, UK) can be visualized using an anti-rabbit immunoglobulinconjugated to horseradish peroxidase (BioRad; Hemel Hempstead, UK) andthe ECL detection system (Amersham, UK). Western blotting resultstypically confirm the presence of the dominant fusion protein togetherwith several faint bands corresponding to proteins of lower MW than thefully sized fusion protein. This observation suggests that limiteddegradation of the fusion protein occurred in the bacteria or during theisolation procedure.

To produce the subcloned light chain, pellets from 1 liter cultures ofbacteria expressing the wild-type Botulinum neurotoxin light chainproteins can be resuspended in column buffer [10 mM Tris-HCl (pH 8.0),200 mM NaCl, 1 mM EGTA and 1 mM DTT] containing 1 mMphenylmethanesulfonyl fluoride (PMSF) and 10 mM benzamidine, and lysedby sonication. The lysates can be cleared by centrifugation at 15,000×gfor 15 minutes at 4° C. Supernatants can be applied to an amyloseaffinity column [2×10 cm, 30 ml resin] (New England BioLabs; Hitchin,UK). Unbound proteins can be washed from the resin with column bufferuntil the eluate is free of protein as judged by a stable absorbancereading at 280 nm. The bound MBP-L chain fusion protein can besubsequently eluted with column buffer containing 10 mM maltose.Fractions containing the fusion protein can be pooled and dialyzedagainst 20 mM Tris-HCl (pH 8.0) supplemented with 150 mM NaCl, 2 mM,CaCl₂ and 1 mM DTT for 72 hours at 4° C.

The MBP-L chain fusion proteins can be purified after release from thehost bacteria. Release from the bacteria can be accomplished byenzymatically degrading or mechanically disrupting the bacterial cellmembrane. Amylose affinity chromatography can be used for purification.Recombinant wild-type or mutant light chains can be separated from thesugar binding domains of the fusion proteins by site-specific cleavagewith Factor Xa. This cleavage procedure typically yields free MBP, freelight chains and a small amount of uncleaved fusion protein. While theresulting light chains present in such mixtures can be shown to possessthe desired activities, an additional purification step can be employed.For example, the mixture of cleavage products can be applied to a secondamylose affinity column which binds both the MBP and uncleaved fusionprotein. Free light chains can be isolated in the flow through fraction.

Example 10 Reconstitution of Native Light Chain, Recombinant Wild-TypeLight Chain with Purified Heavy Chain

Native heavy and light chains can be dissociated from a BoNT with 2 Murea, reduced with 100 mM DTT and then purified according to establishedchromatographic procedures. For example, Kozaki et al. (1981, Japan J.Med. Sci. Biol. 34, 61) and Maisey et al. (1988, Eur. J. Biochem. 177,683). A purified heavy chain can be combined with an equimolar amount ofeither native light chain or a recombinant light chain. Reconstitutioncan be carried out by dialyzing the samples against a buffer consistingof 25 mM Tris (pH 8.0), 50 μM zinc acetate and 150 mM NaCl over 4 daysat 4° C. Following dialysis, the association of the recombinant lightchain and native heavy chain to form disulfide linked 150 kDa dichainsis monitored by SDS-PAGE and/or quantified by densitometric scanning.

Example 11 Production of a Modified Neurotoxin with an EnhancedBiological Persistence

A modified neurotoxin can be produced by employing recombinanttechniques in conjunction with conventional chemical techniques.

A neurotoxin chain, for example a botulinum light chain that is to befused with a biological persistence enhancing component to form amodified neurotoxin can be produced recombinantly and purified asdescribed in example 9.

The recombinant neurotoxin chain derived from the recombinant techniquescan be covalently fused with (or coupled to) a biological persistenceenhancing component, for example a leucine-based motif, a tyrosine-basedmotif and/or an amino acid derivative. Peptide sequences comprisingbiological persistence enhancing components can be synthesized bystandard t-Boc/Fmoc technologies in solution or solid phase as is knownto those skilled in the art. Similar synthesis techniques are alsocovered by the scope of this invention, for example, methodologiesemployed in Milton et al. (1992, Biochemistry 31, 8799-8809) and Swainet al. (1993, Peptide Research 6, 147-154). One or more synthesizedbiological persistence enhancing components can be fused to the lightchain of botulinum type A, B, C1, C2, D, E, F or G at, for example, thecarboxyl terminal end of the toxin. The fusion of the biologicalpersistence enhancing components is achieved by chemical coupling usingreagents and techniques known to those skilled in the art, for examplePDPH/EDAC and Traut's reagent chemistry.

Alternatively, a modified neurotoxin can be produced recombinantlywithout the step of fusing the biological persistence enhancingcomponent to a recombinant botulinum toxin chain. For example, arecombinant neurotoxin chain, for example, a botulinum light chain,derived from the recombinant techniques of example 9 can be producedwith a biological persistence enhancing component, for example aleucine-based motif, a tyrosine-based motif and/or an amino acidderivative. For example, one or more DNA sequences encoding biologicalpersistence enhancing components can be added to the DNA sequenceencoding the light chain of botulinum type A, B, C1, C2, D, E, F or G.This addition can be done by any number of methods used for sitedirected mutagenesis which are familiar to those skilled in the art.

The recombinant modified light chain containing the fused or addedbiological persistence enhancing component can be reconstituted with aheavy chain of a neurotoxin by the method described in example 10thereby producing a complete modified neurotoxin.

The modified neurotoxins produced according to this example have anenhanced biological persistence. Preferably, the biological persistenceis enhanced by about 20% to about 300% relative to an identicalneurotoxin without the additional biological persistence enhancingcomponent(s).

Example 12

The first 30 residues of the amino-terminus (N-term) and the last 50residues of the carboxyl-terminal (C-term) of the amino acid sequencesof botulinum toxin serotypes A through G light chains (LC) are shown inTable 2.

TABLE 2 Toxin N-term (AAs 1-30) of LC SEQ ID NO: C-term (last 50 AAs) ofLC SEQ ID NO: BoNT/A MPFVNKQFNYKDPVNGVDIAY 14 GFNLRNTNLAANFNGQNTEIN 15IKIPNAGQM NMNFTKLKNFTGLFEFYKLLC VRGIITSK BoNT/B MPVTINNFNYNDPIDNDNIIM 16YTIEEGFNISDKNMGKEYRGQ 17 MEPPFARGT NKAINKQAYEEISKEHLAVYK IQMCKSVK BoNT/C₁ MPITINNFNYSDPVDNKNILY 18 NIPKSNLNVLFMGQNLSRNPA 19 LDTHLNTLALRKVNPENMLYLFTKFCHKAI DGRSLYNK BoNT/D MTWPVKDFNYSDPVNDNDILY 20YTIRDGFNLTNKGFNIENSGQ 21 LRIPQNKLI NIERNPALQKLSSESVVDLFT KVCLRLTK BoNT/EMPKINSFNYNDPVNDRTILYI 22 GYNINNLKVNFRGQNANLNPR 23 KPGGCQEFYIITPITGRGLVKKIIRFCKNI VSVKGIRK BoNT/F MPVAINSFNYNDPVNDDTILY 24TVSEGFNIGNLAVNNRGQSIK 25 MQIPYEEKS LNPKIIDSIPDKGLVEKIVKF CKSVIPRK BoNT/GMPVNIKNFNYNDPINNDDIIM 26 QNEGFNIASKNLKTEFNGQNK 27 MEPFNDPGPAVNKEAYEEISLEHLVIYRIA MCKPVMYK

Alterations in the amino acid sequence of these serotypes can includeamino acid substitutions, mutations, deletions, or various combinationsof these alterations. Such alterations can be engineered in the firstthirty amino acids (AAs) in the N-terminus of the light chain and/or thelast fifty AAs in the C-terminus of the light chain using recombinantDNA technological methods that are standard in the art.

For example, studies showed that a GFP-LCA construct with eight aminoacid residues (PFVNKQFN) (SEQ ID NO:120) deleted from the N-terminus (noC-terminus deletion) localized in PC12 cells a very similar pattern tothe localization in PC12 cells of a truncated GFP-LCA construct withboth the C and N terminus deletions.

Further studies showed that a GFP-LCA construct with twenty two aminoacid residues (KNFTG LFEFYKLLCV RGIITSK) (SEQ ID NO:121) deleted fromthe C-terminus (no N-terminus deletion) localized in PC12 cells in avery similar manner to that of the GFP-LCA(LL-->AA) mutant.

A GFP-LCA construct with both eight amino acid residues (PFVNKQFN; SEQID NO:120) deleted from the N-terminus and twenty two amino acidresidues (KNFTG LFEFYKLLCV RGIITSK; SEQ ID NO:121) deleted from theC-terminus accumulated intracellularly.

Examples of amino acid sequence substitutions include the replacement ofone or more contiguous or non-contiguous amino acids in the first 30amino acids of the N-terminus and/or the last 50 amino acids of theC-terminus of the light chain with an equal number and placement ofamino acids that differ from the wild-type sequence. Substitutions canbe conservative or non-conservative of the character of the amino acid.For example, the amino acid valine at a specific position in thewild-type sequence can be replaced with an alanine in the same positionin the substituted sequence. Furthermore, basic residues such asarginine or lysine can be substituted for highly hydrophobic residuessuch as tryptophan. A proline or histidine residue may be substituted inorder to form or disrupt a potentially important structural or catalyticelement of the protein. Some examples of amino acid substitutions areindicated by bold underlined text in the sequences described in Table 3.

TABLE 3 Toxin N-term (AAs 1-30) of LC SEQ ID NO: C-term (last 50 AAs) ofLC SEQ ID NO: BoNT/A MPF A NKQFNYKDPVNGVDIAY 28 GFNLRNTNLAANFNGQNTEIN 29IKIPNAGQM NMN R TKLKNFTGLFEFYKLLC VRGIITSK BoNT/A MPFVNKQFN KKDPVNGVDIAY 30 GFNLRNTNLAANFNGQNTEIN 31 IKIPNAGQM NMNFTKLKN AAGLFEFYKLLC VRGIITSK BoNT/A MPFVNKQFNYKDPVNGVDIA R 32 GFNLRNTNLAAN HNGQNTEIN 33 IKIPNAGQM NMNFTKLKNFTGLFEFYKLLC VRGIITSK BoNT/A MPFVNK HFNYKDPVNGVDIAY 34 GFNLRNTNLAANFNGQNTEIN 35 IKIPNAGQMNMNFTKLKNFTGLFEFYKLLC A RGIITSK BoNT/B MP A TINNFNYNDPIDNDNIIM 36YTIEEGFNISDKNMGKEYRGQ 37 MEPPFARGT NKAINKQAYEEISKEHLAVYK I R MCKSVKBoNT/B MPVTINNFNYNDPIDNDNII A 38 YTIEEGFNISDKNMGKEYRGQ 39 A EPPFARGTNKAINKQAYEEISKEHLAV R K IQMCKSVK BoNT/B MPVTINNFN R NDPIDNDNIIM 40YTIEEGFNISDKNMGKEYRGQ 41 MEPPFARGT NKAINKQA K EEISKEHLAVYK IQMCKSVKBoNT/C ₁ MPITINN K NYSDPVDNKNILY 42 NIPKSNLNVLFMGQNLSRNPA 43 LDTHLNTLALRKVNPENMLYLFTKFCHKAI DGRSL R NK BoNT/D MTWP A KDFNYSDP A NDNDILY 44YTIRDGFNLTNKGFNIENSGQ 45 LRIPQNKLI NIERNPALQKLSSESVVDLFT K A CLRLTKBoNT/E MPKINSFNYNDP A NDRTILYI 46 GYNINNLKVNFRGQNANLNPR 47 KPGGCQEFYIITPITGRG H VKKIIRFCKNI VSVKGIRK BoNT/E MPKINS R NYNDPVNDRTILYI 48GYNINNLKVNFRGQNANLNPR 49 KPGGCQEFY IITPITGRGLVKKIIRFCKN A A SVKGIRKBoNT/E MPKINSFNYNDPVNDRTILYI 50 GYNINNLKVNFRGQNANLNPR 51 KPGGCQEF RIITPITGRGLVKKIIRFCKNI VS A KGIRK BoNT/F MP A AINSFNYNDPVNDDTILY 52TVSEGFNIGNLAVNNRGQSIK 53 MQIPYEEKS LNPKIIDSIPDKGLVEKIVKF CKS A IPRKBoNT/G MPVNIKNH NYNDPINNDDIIM 54 QNEGFNIASKNLKTEFNGQNK 55 MEPFNDPGPAVNKEAYEEISLEHLVIYRIA MCKP A MYK

Examples of amino acid sequence mutations include changes in the first30 amino acids of the N-terminus and/or the last 50 amino acids of theC-terminus of the light chain sequence such that one or several aminoacids are added, substituted and/or deleted such that the identity,number and position of amino acids in the wild-type light chain sequenceis not necessarily conserved in the mutated light chain sequence. Someexamples of amino acid sequence mutations are described in Table 4, inwhich additions of amino acids are shown in bold underlined text, anddeletions are indicated by dashes in the sequences shown.

TABLE 4 Toxin N-term (AAs 1-30) of LC SEQ ID NO: C-term (last 50 AAs) ofLC SEQ ID NO: BoNT/A MPFVNKQFNYKDPVNGVDIAY 56 GFNLRNTNLAANFNGQNTEIN 57IKIP H ---- NMN AAAAAAAAAA ------- CVRGIITSK BoNT/A M AAA ---- 58 G KNLRNTNLAANFNGQNTEIN 59 NYKDPVNGVDIAYIKIPNAGQ NMNFTKLKNFTGLFEFYK- MCVRGIITSK BoNT/A MPFVNKQFNYKDPVNGVDIA R 60 GFNLRNTNLAA---- 61 ----NAGQMH NTEINNMNFTKLKNFTGLFE FYKLLCVRGIITSK BoNT/A MP K VNKQFN---- 62GFNLRNTNLAANFNGQNTEIN 63 VNGVDIAYIKIPNAGQM NMNFTKLKNFTGLFEF RR --------TSK BoNT/B MPVTINNFNYNDPIDNDNII A 64 YTI PP GFNISDKNMGKEYRGQ 65AAAAA ARGT NKAINKQAYEEISKEH----- -------- BoNT/B MP A ---- 66YTIEEGFNISDKNMGKEYRGQ 67 FNYNDPIDNDNIIMMEPPFAR NKA AAAAAA EEISKEHLAVYKGT IQMCKSVK BoNT/B MPVTINNFN R ---------- 68 YTIEEGFNISDKNMGKEYRGQ 69MMEPPFARGT NKAINKQAY------ AAAAAA IQMCKSVK BoNT/C ₁ M--------- 70NIPKSNLNVLFMGQNLSRNPA 71 SDPVDNKNILYLDTHLNTLA LRKVNPENML AAA ---CHKAIDGRSLYNK BoNT/D MT R PVKD---- 72 YTIRDGFNLTNKGFNIENSGQ 73DPVNDNDILYLRIPQNKLI NIERNPALQKL------ DL P PKVCLRLTK BoNT/E MPKINS PPNYNDPVNDRTILY 74 GYNINNLKVNFRGQNANLNPR 75 IKPGGCQEFY IITPITGRGLVKK AAAACKNI VSVKGIRK BoNT/E MPKINSFNYNDP AAAA NDRTI 76 GYNINNLKVNFRGQNANLNPR 77LYIKPGGCQEFY IITPITGRGLV--- H RFCKNIVSVKGIRK BoNT/E MPKINSFNYNDPVNDRTILK I 78 GYNINNLKVNFRGQNANLNPR 79 KPGGC K EFY IITPITGRGL PP ----------------- BoNT/F MP------ 80 TVSEGFNIGNLAVNNRGQSIK 81NYNDPVNDDTILYMQIPYEEK LNPKIIDSIPDKG AAAAAA -- S CKSVIPRK BoNT/G MPVNI PP---- 82 QNEGFNIASKNLKTEFNGQNK 83 DPINNDDIIMMEPFNDPGPAVNKEAY-------------- - AAAAAAA

Examples of amino acid sequence deletions include the removal of one ormore contiguous or non-contiguous amino acids from the first 30 aminoacids of the N-terminus and/or the last 50 amino acids of the C-terminusof the light chain sequence. Some examples of amino acid sequencedeletions are indicated by dashes in the sequences shown in Table 5.

TABLE 5 Toxin N-term (AAs 1-30) of LC SEQ ID NO: C-term (last 50 AAs) ofLC SEQ ID NO: BoNT/A M-------- 84 GFNLRNTNLAANFNGQNTEIN 85YKDPVNGVDIAYIKIPNAGQM NMNFTKLKNFTGLFEFYK--- -------- BoNT/AMPFVNKQ------ 86 GFNLRNTNLAANFNGQNTEIN 87 VNGVDIAYIKIPNAGQMNMNFTKLK---------- LLCVRGIITSK BoNT/A MPFVNKQFNYKDP------ 88GFNLRNTNLAANFNGQNTEIN 89 AYIKIPNAGQM NMN-------- GLFEFYKLLCVRGIITSKBoNT/A MPFVNKQFNYKDPVNGVDIA- 90 GFNLRN---------- 91 ---------NTEINNMNFTKLKNFTGLFEF YKLLCVRGIITSK BoNT/B MPVTINNFNYNDPIDNDNIIM 92YTI----- 93 ME------- ISDKNMGKEYRGQNKAINKQA YEEISKEHLAVYKIQMCKSVK BoNT/BMPVTINNFNYND--------- 94 YTIEEGFNISD-------- 95 -EPPFARGTGQNKAINKQAYEEISKEHLAV YKIQMCKSVK BoNT/B MP-------- 96YTIEEGFNISDKNMGKEYRGQ 97 NDPIDNDNIIMMEPPFARGT NKAINKQA------------KIQMCKSVK BoNT/C ₁ MPI------- 98 NIPKSNLNVLFMGQNLSRNPA 99SDPVDNKNILYLDTHLNTLA LRKV---------- KFCHKAIDGRSLYNK BoNT/D MTW----------100  YTIRDGFNLTNKGFNIENSGQ 101  VNDNDILYLRIPQNKLI NIERNPA----------DLFTKVCLRLTK BoNT/E MP-------- 102  GYNINNLKVNFRGQNANLNPR 103 DPVNDRTILYIKPGGCQEFY IITPI---------- RFCKNIVSVKGIRK BoNT/EMPKINSFNYN---------- 104  GYNINN------ 105  IKPGGCQEFYGQNANLNPRIITPITGRGLVK KIIRFCKNIVSVKGIRK BoNT/E MPKINSFNYNDPVNDRTILYI106  GYNINNLKVNFRGQNANLNPR 107  K-------- IITPITGRGLVKKIIR----- ---KGIRKBoNT/F MPVAINSFNYNDPVNDDTILY 108  TVSEGFNIGNLAVNNRGQSIK 109  MQIP-----LNPKIIDSIPD-------- KFCKSVIPRK BoNT/G M-------------------- —QNEGFNIASKNLKTEFNGQNK 110  --------- AVNKEA------------ RIAMCKPVMYK

Example 13

In some embodiments of the present invention, the biological persistenceand/or the enzymatic activity of a toxin can be altered by structurallymodifying the toxin. In some embodiments, the structural modificationincludes the substitution, mutation or deletion of amino acids withinthe toxin. In some embodiments, the structural modification includes achimeric fusion construct in which a biological persistence-enhancingcomponent or an enzymatic activity-enhancing component may be fused to,swapped for, or incorporated within a terminal end of the light chain ofa botulinum toxin. In some embodiments, the structural modificationincludes a chimeric fusion construct in which a biologicalpersistence-reducing component or an enzymatic activity-reducingcomponent may be fused to, swapped for, or incorporated within aterminal end of the light chain of a botulinum toxin. In someembodiments, the persistence- or activity-enhancing or persistence- oractivity-reducing component is an N-terminal region including the first30 amino acids of a light chain of a botulinum toxin, or a C-terminalregion including the last 50 amino acids of a light chain of a botulinumtoxin. This biological persistence- or enzymatic activity-enhancingcomponent or biological persistence- or enzymatic activity-reducingcomponent is swapped for, fused to, or incorporated within an N- and/orC-terminus of a light chain of a botulinum toxin to enhance or reduceits biological persistence and/or enzymatic activity.

In some embodiments, the fusion of, addition to, or swapping of theN-terminal region of the light chain of BoNT/A into a chimeric constructresults in an increase in biological persistence and/or enzymaticactivity. In some embodiments, a substituted, mutated, or deletedN-terminal region of the light chain of BoNT/A within a chimericconstruct results in a decrease in biological persistence and/orenzymatic activity. In some embodiments, the fusion of, addition to, orswapping of the C-terminal region of the light chain of BoNT/A into achimeric construct results in an increase in biological persistenceand/or enzymatic activity. In some embodiments, a substituted, mutated,or deleted C-terminal region of the light chain of BoNT/A within achimeric construct results in a decrease in biological persistenceand/or enzymatic activity.

Generally, it is suitable that the chimeric toxin has a biologicalpersistence of about 20% to 300% greater than an identical toxin withoutthe structural modification. The biological persistence of the chimerictoxin may be enhanced by about 100%. That is, for example, the modifiedbotulinum neurotoxin including the biological persistence-enhancingcomponent is able to cause a substantial inhibition of neurotransmitterrelease (for example, acetylcholine) from a nerve terminal for about 20%to about 300% longer than a neurotoxin without the structuralmodification.

Similarly, it is suitable that the chimeric botulinum toxin light chainhas an altered enzymatic activity. For example, the chimeric toxin canexhibit a reduced or an enhanced inhibition of exocytosis (such asexocytosis of a neurotransmitter) from a target cell with or without anyalteration in the biological persistence of the modified neurotoxin.Altered enzymatic activities include increased or decreased efficiencyor potency, increased or decreased localization to the plasma membrane,increased or decreased substrate specificity, and/or increased ordecreased rate of proteolysis of SNAP/SNARE proteins. An increase inenzymatic activity can be from 1.5 to 5 times greater than thebiological activity of the native or unmodified light chain. Forexample, the chimeric botulinum neurotoxin including the enzymaticactivity-enhancing component is able to cause a substantial inhibitionof neurotransmitter release (for example, acetylcholine) from a nerveterminal due to an increased rate of proteolysis of the SNAP-25substrate as compared to a neurotoxin without the structuralmodification.

It has been observed that a recombinant construct with both eight aminoacid residues (PFVNKQFN; SEQ ID NO: 120) deleted from the N-terminus andtwenty-two amino acid residues (KNFTG LFEFYKLLCV RGIITSK; SEQ ID NO:121) deleted from the C-terminus of the light chain of botulinum toxin Aexhibits a reduced activity such that the effective concentration (EC₅₀)required to cleave the SNAP-25 substrate is nearly ten-fold greater thanthat of a similar construct with only the C-terminal twenty-two aminoacid deletion (EC₅₀ ΔN8ΔC22=4663 pM vs. EC₅₀Δ C22=566 pM). Therecombinant light chain of botulinum toxin A was used as a control (EC₅₀rLC/A=7 pM), and, therefore, as compared to the rLC/A construct, a666-fold greater concentration of the ΔNΔ8C22 construct is required. Arecombinant light chain construct with the dileucine motif mutated todialanine [rLC/A(LL-->AA)] also exhibits reduced activity (EC₅₀rLC/A(LL-->AA)=184 μM); however, the effective concentration of theΔN8ΔC22 construct is twenty-five fold greater than the rLC/A(LL-->AA)construct.

A modified light chain may include a light chain from botulinum toxinsA, B, C1, D, E, F or G. One or multiple domains at the N- and/orC-terminus may be modified by addition, deletion or substitution. Forexample, a modified chimeric light chain component may include a lightchain from BoNT/E modified by adding or replacing/substituting one ormore N- and/or C-terminal end sequences derived from the BoNT/A lightchain, thereby resulting in a chimeric BoNT/E-BoNT/A chimeric lightchain with one or both terminal ends having one or more sequences whichconvey an increased or decreased ability to localize to a plasmamembrane, increased or decreased biological persistence and/or anincreased or decreased enzymatic activity.

A chimeric botulinum toxin can be constructed such that a C-terminalportion of the light chain of one botulinum toxin serotype replaces asimilar C-terminal portion within the light chain of another botulinumtoxin serotype. For example, the last twenty two amino acid residuesbearing the dileucine motif from the C-terminus of the light chain ofBoNT/A can replace the last twenty two amino acid residues of theC-terminus of the light chain of BoNT/E. The amino acid sequence of theentire light chain of such a chimeric construct is shown below:

(SEQ ID NO: 124) MPKINSFNYNDPVNDRTILYIKPGGCQEFYKSFNIMKNIWIIPERNVIGTTPQDFHPPTSLKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGGILLEELSKANPYLGNDNTPDNQFHIGDASAVEIKFSNGSQDILLPNVIIMGAEPDLFETNSSNISLRNNYMPSNHGFGSIAIVTFSPEYSFRFNDNSMNEFIQDPALTLMHELIHSLHGLYGAKGITTKYTITQKQNPLITNIRGTNIEEFLTFGGTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYKDVFEAKYGLDKDASGIYSVNINKFNDIFKKLYSFTEFDLATKFQVKCRQTYIGQYKYFKLSNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIITPIT GKNFTGLFEFYKLLCVRGIITSK

In the construct above, the majority of the amino acid sequence isderived from BoNT/E serotype, and the amino acids shown in boldunderlined text are derived from the last twenty two amino acid residuesof the C-terminus of the light chain of BoNT/A which bears the dileucinemotif.

In a further example, the first thirty amino acid residues from theN-terminus of the light chain of BoNT/A can replace the first thirtyamino acid residues of the N-terminus of the light chain of BoNT/B. Theamino acid sequence of the entire light chain of such a chimericconstruct is shown below:

(SEQ ID NO: 125) MPFVNKQFNYKDPVNGVDIAYIKIPNAGQM GRYYKAFKITDRIWIIPERYTFGYKPEDFNKSSGIFNRDVCEYYDPDYLNTNDKKNIFFQTLIKLFNRIKSKPLGEKLLEMIINGIPYLGDRRVPLEEFNTNIASVTVNKLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNHFASREGFGGIMQMKFCPEYVSVFNNVQENKGASIFNRRGYFSDPALILMHELIHVLHGLYGIKVDDLPIVPNEKKFFMQSTDTIQAEELYTFGGQDPSIISPSTDKSIYDKVLQNFRGIVDRLNKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFNKLYKSLMLGFTEINIAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNISDKNMGKEYRGQNKAINKQAYEEISKEHLAVYKIQMCKSVK

In the construct above, the majority of the amino acid sequence isderived from BoNT/B serotype, and the amino acids shown in boldunderlined text are derived from the first thirty amino acid residues ofthe N-terminus of the light chain of BoNT/A.

Still further, the chimeric construct can have both N-terminal and theC-terminal replacements. For example, the first nine amino acid residuesfrom the N-terminus of the light chain of BoNT/A can replace the firstnine amino acid residues of the N-terminus of the light chain of BoNT/E.Additionally, in the same construct, the last twenty-two amino acidresidues from the C-terminus of the light chain of BoNT/A can replacethe last twenty-two amino acid residues from the C-terminus of the lightchain of BoNT/E. The amino acid sequence of the entire light chain ofsuch a chimeric construct is shown below:

(SEQ ID NO: 126) MPFVNKQFN NDPVNDRTILYIKPGGCQEFYKSFNIMKNIWIIPERNVIGTTPQDFHPPTSLKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGGILLEELSKANPYLGNDNTPDNQFHIGDASAVEIKFSNGSQDILLPNVIIMGAEPDLFETNSSNISLRNNYMPSNHGFGSIAIVTFSPEYSFRFNDNSMNEFIQDPALTLMHELIHSLHGLYGAKGITTKYTITQKQNPLITNIRGTNIEEFLTFGGTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYKDVFEAKYGLDKDASGIYSVNINKFNDIFKKLYSFTEFDLATKFQVKCRQTYIGQYKYFKLSNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIITPIT GKNFTGLFEFYKLLCVRGIITSK

In the construct above, the majority of the amino acid sequence isderived from BoNT/E serotype, and the amino acids shown in boldunderlined text are derived from the first nine amino acid residues ofthe N-terminus and the last twenty-two amino acid residues of theC-terminus of the light chain of BoNT/A.

Similarly, the first nine amino acid residues from the N-terminus of thelight chain of BoNT/A can replace the first nine amino acid residues ofthe N-terminus of the light chain of BoNT/B. Additionally, in the sameconstruct, the last twenty-two amino acid residues from the C-terminusof the light chain of BoNT/A can replace the last twenty-two amino acidresidues from the C-terminus of the light chain of BoNT/B. The aminoacid sequence of the entire light chain of such a chimeric construct isshown below:

(SEQ ID NO: 127) MPFVNKQFN YNDPIDNDNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFNKSSGIFNRDVCEYYDPDYLNTNDKKNIFFQTLIKLFNRIKSKPLGEKLLEMIINGIPYLGDRRVPLEEFNTNIASVTVNKLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNHFASREGFGGIMQMKFCPEYVSVFNNVQENKGASIFNRRGYFSDPALILMHELIHVLHGLYGIKVDDLPIVPNEKKFFMQSTDTIQAEELYTFGGQDPSIISPSTDKSIYDKVLQNFRGIVDRLNKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFNKLYKSLMLGFTEINIAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNI SDKNMGKEYRGQNKAINKQKNFTGLFEFYKLLCVRGIITSK

In the construct above, the majority of the amino acid sequence isderived from BoNT/B serotype, and the amino acids shown in boldunderlined text are derived from the first nine amino acid residues ofthe N-terminus and the last twenty-two amino acid residues of theC-terminus of the light chain of BoNT/A.

Furthermore, the first nine amino acid residues from the N-terminus ofthe light chain of BoNT/A can replace the first nine amino acid residuesof the N-terminus of the light chain of BoNT/F. Additionally, in thesame construct, the last twenty-two amino acid residues from theC-terminus of the light chain of BoNT/A can replace the last twenty-twoamino acid residues from the C-terminus of the light chain of BoNT/F.The amino acid sequence of the entire light chain of such a chimericconstruct is shown below:

(SEQ ID NO: 128) MPFVNKQFN YNDPVNDDTILYMQIPYEEKSKKYYKAFEIMRNVWIIPERNTIGTNPSDFDPPASLKNGSSAYYDPNYLTTDAEKDRYLKTTIKLFKRINSNPAGKVLLQEISYAKPYLGNDHTPIDEFSPVTRTTSVNIKLSTNVESSMLLNLLVLGAGPDIFESCCYPVRKLIDPDVVYDPSNYGFGSINIVTFSPEYEYTFNDISGGHNSSTESFIADPAISLAHELIHALHGLYGARGVTYEETIEVKQAPLMIAEKPIRLEEFLTFGGQDLNIITSAMKEKIYNNLLANYEKIATRLSEVNSAPPEYDINEYKDYFQWKYGLDKNADGSYTVNENKFNEIYKKLYSFTESDLANKFKVKCRNTYFIKYEFLKVPNLLDDDIYTVSEGFNIGNLAVN NRGQSIKLNPKIIDKNFTGLFEFYKLLCVRGIITSK

In the construct above, the majority of the amino acid sequence isderived from BoNT/F serotype, and the amino acids shown in boldunderlined text are derived from the first nine amino acid residues ofthe N-terminus and the last twenty-two amino acid residues of theC-terminus of the light chain of BoNT/A.

In some embodiments, a light chain can be engineered such that one ormore segments of the light chain of one or more toxin serotypes replaceone or more segments of equal or unequal length within the light chainof another toxin serotype. In a non-limiting example of this kind ofchimeric construct, fifty amino acid residues from the N-terminus of thelight chain of BoNT/A can replace eight amino acid residues of theN-terminus of the light chain of BoNT/B, resulting in a net gain offorty-two amino acids in length in the N-terminal region of the lightchain chimera. The amino acid sequence of the entire light chain of sucha chimeric construct is shown below:

(SEQ ID NO: 129) M PFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDT FYNDPIDNDNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFNKSSGIFNRDVCEYYDPDYLNTNDKKNIFFQTLIKLFNRIKSKPLGEKLLEMIINGIPYLGDRRVPLEEFNTNIASVTVNKLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNHFASREGFGGIMQMKFCPEYVSVFNNVQENKGASIFNRRGYFSDPALILMHELIHVLHGLYGIKVDDLPIVPNEKKFFMQSTDTIQAEELYTFGGQDPSIISPSTDKSIYDKVLQNFRGIVDRLNKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFNKLYKSLMLGFTEINIAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNISDKNMGKEYRGQNKAINKQAYEEISKEHLAVYKIQMCKSVK

In the construct above, the majority of the amino acid sequence isderived from BoNT/B serotype, and the amino acids shown in boldunderlined text are derived from the first fifty amino acid residues ofthe N-terminus of the light chain of BoNT/A.

In a non-limiting example of this kind of chimeric construct, the lastfifty amino acid residues from the C-terminus of the light chain ofBoNT/A can replace fifteen amino acid residues within the C-terminus ofthe light chain of BoNT/E, resulting in a net gain of thirty-five aminoacids in the C-terminal region of the light chain chimera. The aminoacid sequence of the entire light chain of such a chimeric construct isshown below:

(SEQ ID NO: 130) MPKINSFNYNDPVNDRTILYIKPGGCQEFYKSFNIMKNIWIIPERNVIGTTPQDFHPPTSLKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGGILLEELSKANPYLGNDNTPDNQFHIGDASAVEIKFSNGSQDILLPNVIIMGAEPDLFETNSSNISLRNNYMPSNHGFGSIAIVTFSPEYSFRFNDNSMNEFIQDPALTLMHELIHSLHGLYGAKGITTKYTITQKQNPLITNIRGTNIEEFLTFGGTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYKDVFEAKYGLDKDASGIYSVNINKFNDIFKKLYSFTEFDLATKFQVKCRQTYIGQYKYFKLSNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIITP GFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSK NI VSVKGIRK

In the construct above, the majority of the amino acid sequence isderived from BoNT/E serotype, and the amino acids shown in boldunderlined text are derived from the last fifty amino acid residues ofthe C-terminus of the light chain of BoNT/A.

In a non-limiting example of this kind of chimeric construct, thirtyamino acid residues from the N-terminus of the light chain of BoNT/A canreplace ten amino acid residues of the N-terminus of the light chain ofBoNT/E, resulting in a net gain of twenty amino acids in length in theN-terminal region of the chimera. Additionally, in the same construct,the last fifty amino acid residues from the C-terminus of the lightchain of BoNT/A can replace the last fifty amino acid residues from theC-terminus of the light chain of BoNT/E. The amino acid sequence of theentire light chain of such a chimeric construct is shown below:

(SEQ ID NO: 131) MPKINSFNY MPFVNKQFNYKDPVNGVDIAYIKIPNAGQM YIKPGGCQEFYKSFNIMKNIWIIPERNVIGTTPQDFHPPTSLKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGGILLEELSKANPYLGNDNTPDNQFHIGDASAVEIKFSNGSQDILLPNVIIMGAEPDLFETNSSNISLRNNYMPSNHGFGSIAIVTFSPEYSFRFNDNSMNEFIQDPALTLMHELIHSLHGLYGAKGITTKYTITQKQNPLITNIRGTNIEEFLTFGGTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYKDVFEAKYGLDKDASGIYSVNINKFNDIFKKLYSFTEFDLATKFQVKCRQTYIGQYKYFKLSNLLNDSIYNISE GFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSK

In the construct above, the majority of the amino acid sequence isderived from BoNT/E serotype, and the amino acids shown in boldunderlined text are derived from the thirty amino acid residues of theN-terminus and the last fifty amino acid residues of the C-terminus ofthe light chain of BoNT/A.

In a non-limiting example of this kind of chimeric construct, thirtyamino acid residues from the N-terminus of the light chain of BoNT/A canreplace ten amino acid residues of the N-terminus of the light chain ofBoNT/B, resulting in a net gain of twenty amino acids in length in theN-terminal region of the chimera. Additionally, in the same construct,the last fifty amino acid residues from the C-terminus of the lightchain of BoNT/A can replace the last fifty amino acid residues from theC-terminus of the light chain of BoNT/B. The amino acid sequence of theentire light chain of such a chimeric construct is shown below:

(SEQ ID NO: 132) MPVTINNFN MPFVNKQFNYKDPVNGVDIAYIKIPNAGQM IMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFNKSSGIFNRDVCEYYDPDYLNTNDKKNIFFQTLIKLFNRIKSKPLGEKLLEMIINGIPYLGDRRVPLEEFNTNIASVTVNKLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNHFASREGFGGIMQMKFCPEYVSVFNNVQENKGASIFNRRGYFSDPALILMHELIHVLHGLYGIKVDDLPIVPNEKKFFMQSTDTIQAEELYTFGGQDPSIISPSTDKSIYDKVLQNFRGIVDRLNKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFNKLYKSLMLGFTEINIAENYKIKTRASYFSDSLPP VKIKNLLDNEIGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYK LLCVRGIITSK

In the construct above, the majority of the amino acid sequence isderived from BoNT/B serotype, and the amino acids shown in boldunderlined text are derived from the thirty amino acid residues of theN-terminus and the last fifty amino acid residues of the C-terminus ofthe light chain of BoNT/A.

In a non-limiting example of this kind of chimeric construct, thirtyamino acid residues from the N-terminus of the light chain of BoNT/A canreplace ten amino acid residues of the N-terminus of the light chain ofBoNT/F, resulting in a net gain of twenty amino acids in length in theN-terminal region of the chimera. Additionally, in the same construct,the last fifty amino acid residues from the C-terminus of the lightchain of BoNT/A can replace the last fifty amino acid residues from theC-terminus of the light chain of BoNT/F. The amino acid sequence of theentire light chain of such a chimeric construct is shown below:

(SEQ ID NO: 133) MPVAINSFN MPFVNKQFNYKDPVNGVDIAYIKIPNAGQM LYMQIPYEEKSKKYYKAFEIMRNVWIIPERNTIGTNPSDFDPPASLKNGSSAYYDPNYLTTDAEKDRYLKTTIKLFKRINSNPAGKVLLQEISYAKPYLGNDHTPIDEFSPVTRTTSVNIKLSTNVESSMLLNLLVLGAGPDIFESCCYPVRKLIDPDVVYDPSNYGFGSINIVTFSPEYEYTFNDISGGHNSSTESFIADPAISLAHELIHALHGLYGARGVTYEETIEVKQAPLMIAEKPIRLEEFLTFGGQDLNIITSAMKEKIYNNLLANYEKIATRLSEVNSAPPEYDINEYKDYFQWKYGLDKNADGSYTVNENKFNEIYKKLYSFTESDLANKFKVKCRNTYFIKYEFLKVPNL LDDDIYGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVR GIITSK

In the construct above, the majority of the amino acid sequence isderived from BoNT/F serotype, and the amino acids shown in boldunderlined text are derived from the thirty amino acid residues of theN-terminus and the last fifty amino acid residues of the C-terminus ofthe light chain of BoNT/A.

In some embodiments, the swapped sequences can be derived from twodifferent serotypes, resulting in a chimera with regions from threedifferent serotypes in all. In this example, eight amino acid residuesfrom the N-terminus of the light chain of BoNT/B can replace five aminoacid residues of the N-terminus of the light chain of BoNT/E, resultingin a net gain of three amino acids in length in the N-terminal region ofthe chimera. Additionally, in the same construct, 30 amino acid residuesincluding the dileucine repeat of the C-terminus of the light chain ofBoNT/A can replace ten amino acid residues within the C-terminus of thelight chain of BoNT/E, resulting in a net gain of 20 amino acids in theC-terminal region of the chimera. The amino acid sequence of the entirelight chain of such a chimeric construct is shown below:

(SEQ ID NO: 134) MPKINSFNYNDP

DRTILYIKPGGCQEFYKSFNIMKNIWIIPERNVIGTTPQDFHPPTSLKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGGILLEELSKANPYLGNDNTPDNQFHIGDASAVEIKFSNGSQDILLPNVIIMGAEPDLFETNSSNISLRNNYMPSNHGFGSIAIVTFSPEYSFRFNDNSMNEFIQDPALTLMHELIHSLHGLYGAKGITTKYTITQKQNPLITNIRGTNIEEFLTFGGTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYKDVFEAKYGLDKDASGIYSVNINKFNDIFKKLYSFTEFDLATKFQVKCRQTYIGQYKYFKLSNLLNDSIYNISEGYNINNLKVNFRGQNANLNPR IITPITGRGLVKKIIRFCKNNMNFTKLKNFTGLFEFYKLLCVRGIITSK

In the construct above, the majority of the amino acid sequence isderived from BoNT/E serotype, and the amino acids shown in bolditalicized text are derived from eight amino acid residues of theN-terminus of the light chain of BoNT/B and thirty amino acid residuesshown in bold underlined text are derived from thirty amino acidresidues of the C-terminus of the light chain of BoNT/A.

In a non-limiting example, eight amino acid residues from the N-terminusof the light chain of BoNT/B can replace five amino acid residues of theN-terminus of the light chain of BoNT/F, resulting in a net gain ofthree amino acids in length in the N-terminal region of the chimera.Additionally, in the same construct, 30 amino acid residues includingthe dileucine repeat of the C-terminus of the light chain of BoNT/A canreplace ten amino acid residues within the C-terminus of the light chainof BoNT/F, resulting in a net gain of 20 amino acids in the C-terminalregion of the chimera. The amino acid sequence of the entire light chainof such a chimeric construct is shown below:

(SEQ ID NO: 135) MPVAINSFNYND

TILYMQIPYEEKSKKYYKAFEIMRNVWIIPERNTIGTNPSDFDPPASLKNGSSAYYDPNYLTTDAEKDRYLKTTIKLFKRINSNPAGKVLLQEISYAKPYLGNDHTPIDEFSPVTRTTSVNIKLSTNVESSMLLNLLVLGAGPDIFESCCYPVRKLIDPDVVYDPSNYGFGSINIVTFSPEYEYTFNDISGGHNSSTESFIADPAISLAHELIHALHGLYGARGVTYEETIEVKQAPLMIAEKPIRLEEFLTFGGQDLNIITSAMKEKIYNNLLANYEKIATRLSEVNSAPPEYDINEYKDYFQWKYGLDKNADGSYTVNENKFNEIYKKLYSFTESDLANKFKVKCRNTYFIKYEFLKVPNLLDDDIYTVSEGFNIGNLAVNNRGQSIKLNPKIIDSIPDKGLVEK NNMNFTKLKNFTGLFEFYKLLCV RGIITSK RK

In the construct above, the majority of the amino acid sequence isderived from BoNT/F serotype, and the amino acids shown in bolditalicized text are derived from eight amino acid residues of theN-terminus of the light chain of BoNT/B and thirty amino acid residuesshown in bold underlined text are derived from thirty amino acidresidues of the C-terminus of the light chain of BoNT/A.

Example 14

The invention also provides for a light chain of a botulinum toxin B,C1, D, E, F or G comprising about the first 30 amino acids from theN-terminus of the light chain of botulinum toxin type A and about thelast 50 amino acids from the C-terminus of the light chain of botulinumtoxin type A. The first 30 amino acids of the N-terminus of type A heremay be all or part, for example 2-16 contiguous or non contiguous aminoacids, of the 30 amino acids. The last 50 amino acids here may be all orpart, for example 5-43 contiguous or non-contiguous, amino acids of the50 amino acids.

In some embodiments, such a light chain comprises about the first 20amino acids from the N-terminus of the light chain of botulinum toxintype A and about the last 30 amino acids from the C-terminus of thelight chain of botulinum toxin type A. The first 20 amino acids of theN-terminus of type A here may be all or part, for example 2-16contiguous or non contiguous amino acids, of the 20 amino acids. Thelast 30 amino acids here may be all or part, for example 5-23 contiguousor non-contiguous, amino acids of the 30 amino acids.

In some embodiments, such a light chain comprises about the first 4 to8, e.g. the first 8, amino acids from the N-terminus of the light chainof botulinum toxin type A and about the last 7 to 22, e.g. the last 22,amino acids from the C-terminus of the light chain of botulinum toxintype A. The first 8 amino acids of the N-terminus of type A here may beall or part, for example 2-7 contiguous or non contiguous amino acids,of the 7 amino acids. The last 22 amino acids here may be all or part,for example 5-16 contiguous or non-contiguous, amino acids of the 20amino acids.

In some embodiments, the inclusion of about the first 30 amino acidsfrom the N-terminus and about the last 50 amino acids from theC-terminus of the light chain of type A replaces one or more amino acidsat the N- and C-termini, respectively, of the light chain of botulinumtoxin type B, C1, D, E, F or G. The first 30 amino acids of theN-terminus of type A here may be all or part, for example 2-16contiguous or non contiguous amino acids, of the 30 amino acids. Thelast 50 amino acids here may be all or part, for example 5-43 contiguousor non-contiguous, amino acids of the 50 amino acids.

In some embodiments, the inclusion of about the 20 amino acids from theN-terminus and about the 30 amino acids from the C-terminus of the lightchain of type A replaces one or more amino acids at the N- andC-termini, respectively, of the light chain of botulinum toxin type B,C1, D, E, F or G. The first 20 amino acids of the N-terminus of type Ahere may be all or part, for example 2-16 contiguous or non contiguousamino acids, of the 20 amino acids. The last 30 amino acids here may beall or part, for example 5-23 contiguous or non-contiguous, amino acidsof the 30 amino acids.

In some embodiments, the inclusion of about the first 4 to 8, forexample the first 8, amino acids from the N-terminus and about the last7 to 22, for example the last 22, amino acids from the C-terminus of thelight chain of type A replaces one or more amino acids at the N- andC-termini, respectively, of the light chain of botulinum toxin type B,C1, D, E, F or G. The first 8 amino acids of the N-terminus of type Ahere may be all or part, for example 2-7 contiguous or non contiguousamino acids, of the 7 amino acids. The last 22 amino acids here may beall or part, for example 5-16 contiguous or non-contiguous, amino acidsof the 20 amino acids.

The invention also provides for a modified botulinum toxin comprisingthe light chain of described herein, including the ones described in theExamples above.

Example 15 Generation of LC/E Chimeras

Truncation of 8 amino acids at the N-terminus of the LC/A (SEQ ID NO:120) completely disrupts plasma membrane localization. The LC/A(ΔN8) iscytoplasmic with a distribution similar to LC/E. The sequences at theN-terminus of LC/A and LC/E are different (FIG. 1).

To generate the LC/E with the N-terminus of the LC/A we pursued twodifferent approaches. The first one was to perform PCR on the nativebeluga LC/E gene with a 5′ primer that contains the N-terminus of theLC/A. Primers for PCR were:

N-ter LC/A forward: (SEQ ID NO: 146)5′ ACCGGATCCCCATTTGTTAATAAACAGTTTAATTATAATGA 3′ N-ter LC/A reverse: (SEQID NO: 147) 5′ CGCGAAGCTTCCTTATGCCTTTTACAGAAACAATATTTTTAC 3′

The PCR was performed with 0.4 μg of the plasmid templatepQBI25fC3beluga LC/E, and 125 ng of each primer. The cycling programwas:

Denaturalizat: 95° C. for 15 min

-   -   5 cycles: 94° C. for 30 sec        -   50° C. for 30 sec        -   72° C. for 1 min    -   25 cycles: 94° C. for 30 sec        -   68° C. for 30 sec        -   72° C. for 1 min

Extension: 72° C. for 10 min

The first five cycles at low annealing temperature will allow the primerto anneal to the 5′ sequence of the LC/E despite the differences insequence. The second set of 25 cycles will use the product with theright sequence for further and more restricted amplification.

The second strategy to generate the LC/E chimera with the N-terminus ofthe LC/A was to mutate one amino acid as a time using site-directedmutagenesis. The primers designed were as follow:

1. Change the sequence on LC/E from Pro-Lys-Ile to Pro-Phe-Val: (SEQ IDNO: 148) 5′ CCGGTTACCGGTACCGGATCCCCATTTGTTAATAGTTTTAATTA T 3′ 2. Changethe sequence Pro-Phe-Val-Asn-Ser to Pro- Phe-Val-Asn-Lys: (SEQ ID NO:149) 5′ CGGATCCCCATTTGTTAATAAATTTAATTATAATGATCCTGTT 3′ 3. Insert aglutamine to complete the N-terminal sequence of the LC/A in the LC/E:(SEQ ID NO: 150) 5′ GATCCCCATTTCTTAATAAACAGTTTAATTATAATGATCCTGTT 3′

Primers shown are on the sense strand, a complimentary primercorresponding to the antisense strand was also ordered and used in thePCR. The template used in the QuikChange mutagenesis was pQBI25fC3belugaLC/E.

We also analyzed the importance of the di-leucine motif present only inthe LC/A by generating a motif in the LC/E in a similar area at theC-terminus (FIG. 2). The sequence in the LC/E reads LxxxII (SEQ ID NO:163). Since Isoleucines can substitute for Leucines in some of the motifpublished we designed primers to generate ExxxII (SEQ ID NO: 144), andalso primers to generate ExxxLL (SEQ ID NO: 145). Those mutations weredone in the native LC/E gene and also in the LC/E containing theN-terminus of the LC/A.

We generated a total of 5 chimeras with all the combinations of theN-terminus of the LC/A and various stages of the di-leucine motifconstruction as seen in FIG. 3. The complete DNA and annotated aminoacid sequence of the wild type LC/E, the chimeric LC/E with theN-terminus of the LC/A, the chimeric LC/E with the LC/A di-leucine motifat the C-terminus, and the full chimeric LC/E with both the N-terminusand the di-leucine motif from LC/A are shown in FIGS. 14-17. We havetested all those mutants for expression, activity, and subcellularlocalization.

Cell Lines and Growth Conditions

SH-SY5Y (Human Neuroblastoma cell line) cells were cultured in Costarbrand polystyrene flasks with vented caps. Growth media consisted ofMinimum Essential Medium with Earle's salts and L-glutamine, F-12Nutrient Mixture (Ham) with L-glutamine, 10% Fetal Bovine Serum(heat-inactivated), Non-Essential Amino-Acids, HEPES, L-Glutamine,Penicillin/Streptomycin. PC-12 (Rat Chromaffin Pheochromocytoma cellline) cells were maintained on Collagen IV coated BD Biocoat dishes (BDBiosciences, Bedford, Mass.). Growth media consisted of RPMI-1640 withL-glutamine, 10% Horse Serum (heat-inactivated), 5% Fetal Bovine Serum(heat-inactivated), HEPES, D-Glucose (Sigma), Sodium Pyruvate,Penicillin/Streptomycin. All (except D-Glucose, Sigma) growth mediacomponents were Gibco products purchased from Invitrogen and all celllines were cultured and maintained at 37° C. with 7.5% CO₂.

Transient Transfection:

The day before transfection, SH-SY5Y cells were plated at 1×10⁶ into6-well plates. Transfections were carried out by diluting LipofectAmine2000 (Invitrogen, Carlsbad, Calif.) at 60 μl per ml in OPTI-MEM ReducedSerum Medium following incubation at RT for 5 min. Next, we diluted 20μg DNA per ml in OPTI-MEM, then combined equal amount of DNA mix andLipofectAmine 2000 (LF) mix, and incubated at RT for another 20 min.Meanwhile, the culture medium in the plates were replaced with 2 ml ofserum free medium, 0.5 ml of DNA+LF mix was then added into the wellsand incubated at 37° C. CO₂ incubator for 6 hours. After the incubationthe medium was removed and replaced with 10% FCS culture medium. Thecells were harvested 24 hours post transfection for further analysis.For confocal analysis, transfections were usually performed in 2-wellculture slices with proportionally reduced amount of reagents.

Transfection into PC12 cells were performed as follow: PC12 cells wereplated the day before transfection in Collagen IV coated dishes at10×10⁶ cells per 100 mm dishes and 2×10⁶ cells per 60 mm dishes. Plateswere transfected with 20 μg or 10 μg respectively with Lipofectamine2000 in OPTI-MEM media. 48 hours after transfection, cells were placedon differentiation media and collected for western blots or fixed in 4%paraformaldehyde for confocal imaging.

Western Blot Analysis:

Cells were collected in 15 ml Falcon tubes, washed once with 1 ml ofPBS, and then transferred to 1.5 ml microcentrifuge tubes. Cells werelysed in 0.5 ml of lysis buffer (50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl₂,1 mM EGTA, 10% glycerol and 1% triton X-100) on rotator at 4° C. for 1hour. Lysed cells were spun down at 5000 rpm at 4° C. for 10 min toeliminate debris; supernatants were transferred to fresh siliconizedtubes. Protein concentrations were measured by the Bradford's method andresuspended in 1×SDS sample buffer at 1 mg/ml or higher concentration.Samples were boiled for 5 min, 20 to 40 μl of the samples were loaded on4-12% Tris-HCl gels. Proteins were transferred to PVDF membranes, andblocked in 5% non-fat milk in TBST buffer for 1 hour at roomtemperature. The cleaved SNAP25 was detected with antiSNAP25₁₉₇ antibodyor with antiSNAP25₁₈₀ diluted in blocking buffer; blot was washedextensively, and the bound antibody was detected with horseradishperoxidase conjugated to species-specific antibody.

When we needed to detect SNAP25₂₀₆, SNAP25₁₉₇ and SNAP25₂₀₆ in the sameblot using the Monoclonal antibody SMI-81 to the N-terminus of theSNAP25, cell lysates were run on a 12% Bis-Tris gel to allow separationof the cleaved SNAP25.

We used the Typhoon 9410 Imager (Amersham) for Western Blot Analysisinstead of traditional film. After the final washes the membrane wasreacted with ECL Plus western blot detection reagent (Amersham) ratherthan SuperSignal reagent used previously, blot was incubated at roomtemperature for 5 min to develop. The choice of pixel size and PMTvoltage settings will depend on the individual blot. Membranes werescanned and quantified using Typhoon Scanner and Imager Analysissoftware.

SH-SY5Y Transient Transfections for SNAP-25 Immunocytochemistry

One day before transfection, SH-SY5Y cells were plated in 60 mm tissueculture dishes at densities of 1.5×10⁶ or 1.6×10⁶ cells per dish toachieve 90-95% confluence at the time of transfection. Transfectionswere performed by diluting 25 μl of Lipofectamine™ 2000 (Invitrogen) in0.5 ml Opti-MEM® I Reduced Serum Medium (Invitrogen) followed byincubation at room temperature for 5 min. DNA (10 μg) was diluted in 0.5ml Opti-MEM® I Reduced Serum Medium. The diluted DNA was mixed gentlywith the diluted Lipofectamine™ 2000. This mixture was incubated for 20min at room temperature. Meanwhile, the culture medium in the plates wasreplaced with 2 ml of serum-free and antibiotic-free medium. The DNAplus Lipofectamine™ 2000 complex was added drop-wise to the cells andmixed into the culture medium by rocking the plates back and forth.Cells were incubated at 37° C., 7.5% CO₂ for 24 hours. Transfectionefficiencies were determined by viewing cells with the fluorescencemicroscope. Transfection efficiencies obtained with the GFP constructwere ca. 40-50%. Lower transfection efficiencies were observed with theGFP-LCE constructs, ca. 10-15%. Antibiotic selection of cells wasaccomplished in complete medium containing 0.5 mg/ml Geneticin G418(Invitrogen) for 48 hours before proceeding with theimmunocytochemistry.

PC-12 Transient Transfections for SNAP-25 Immunocytochemistry

Cells were plated one day before transfection in Collagen IV coateddishes (BD Biosciences) at 1-2×10⁶ cells per 60 mm dish. Plates weretransfected using 10 μg DNA and 25 μl Lipofectamine™ 2000 (each dilutedin 0.5 ml Opti-MEM® I Reduced Serum Medium). The cells were incubatedwith the DNA/Lipofectamine™ 2000 complex for 24 hours in serum- andantibiotic-free medium at 37° C., 7.5% CO₂. The transfection medium wasreplaced with complete growth medium (serum and antibiotics included)containing 0.5 mg/ml G418 (antibiotic selection) and incubationcontinued at 37° C., 7.5% CO₂ for a further 48 hours. Cells were placedin differentiation medium (RPMI-1640 with L-glutamine, D-Glucose(Sigma), Sodium Pyruvate, Penicillin/Streptomycin, BSA (ALBUMAX II,lipid rich), N2-Supplement) containing Nerve Growth Factor (NGF) (HarlanBioproducts for Science, Indianapolis, Ind.) at 50 ng/ml finalconcentration for 24 hours and stained with antibodies specific for GFP,SNAP25₂₀₆ and SNAP25₁₈₀ (Table 6).

TABLE 6 List of antibodies used in the immunocytochemistry experimentsAntibody Source Dilution Used Specificity/Immunogen 1A3A7, ascitesIgG1-K, Allergan 1:100, 1:50 Anti-SNAP25₁₈₀ cleavage mouse monoclonalproduct. Does not cross-react with SNAP25₂₀₆ 1G8C11, ascites IgG1-K,Allergan 1:100, 1:50 Anti-SNAP25₁₈₀ cleavage mouse monoclonal product.Does not cross-react with SNAP25₂₀₆ 1C9F3, ascites IgG1-K, Allergan1:100, 1:50 Anti-SNAP25₁₈₀ cleavage mouse monoclonal product. Does notcross-react with SNAP25₂₀₆ Anti-GFP(FL), rabbitpolyclonal Santa Cruz1:100 Full length GFP (amino acids 1-238 IgG Biotechnologies of AequoreaVictoria origin) Anti-GFP, mouse monoclonal Abcam Inc. 1:100 Full lengthGFP (amino acids 1-246 IgG2 of Aequorea Victoria origin) Anti-SNAP-25,rabbit polyclonal Stressgen 1:100 Amino acids 195-206 of mouse/Biotechnologies human/chicken SNAP-25. Specific for SNAP25₂₀₆ AlexaFluor 568 Goat Anti- Molecular 1:100 Secondary for SNAP-25₂₀₆ Rabbit IgG(H + L), highly cross- Probes detection absorbed Alexa Fluor 488 GoatAnti- Molecular 1:100, 1:200 Secondary for rabbit polyclonal to RabbitIgG (H + L) Probes GFP(FL) Alexa Fluor 568 Goat Anti- Molecular 1:100Secondary for mouse Mouse IgG (H + L) Probes monoclonals to SNAP25₁₈₀Alexa Fluor 488 Goat Anti- Molecular 1:100 Secondary for mousemonoclonal Mouse IgG (H + L) Probes to GFP Immunocytochemistry usingFixative Paraformaldehyde for SNAP25₂₀₆ and SNAP25₁₈₀

Growth medium was removed from cells by aspiration and cells were washedtwice with PBS (Invitrogen, Carlsbad, Calif.). Cells were fixed with 4%paraformaldehyde in PBS (Electron Microscopy Sciences, Washington, Pa.)for 15 to 30 min at room temperature and washed in three changes of PBS.Cells were permeabilized with 0.5% Triton X-100 in PBS for 5 min at roomtemperature and washed in PBS a total of three times. Cells were againpermeabilized in ice-cold methanol for 6 min at −20° C. Methanol wasremoved by aspiration and dishes inverted to allow the cells to dry atroom temperature. Wells were drawn around cells using a Pap pen (Zymed,San Francisco, Calif.) and cells were washed and rehydrated in sixchanges of PBS. Cells were blocked with 100 mM glycine in PBS for 30 minat room temperature followed by three washes in PBS. Cells wereincubated in 0.5% BSA in PBS for 30 min at room temperature washed inthree changes of PBS before addition of the primary antibodies dilutedin 0.5% BSA in PBS (Table 6). Cells were incubated at room temperaturein a humid chamber for 2 hours or at 4° C. overnight. Primary antibodywas removed by a PBS wash without incubation and was followed by three 5min washes in PBS. Cells were incubated with the fluorescently labeledsecondary antibodies (Alexa Fluor Anti-Mouse or Anti-Rabbit Antibodies,Molecular Probes, Table 6) diluted in 0.5% BSA/PBS for 1 hour at roomtemperature in a humid chamber and washed in PBS. Cells were mountedusing VECTASHIELD® Mounting Medium (Vector, Burlingham, Calif.) andcoverslipped. Cells were stored at 4° C. before viewing with a Leicaconfocal microscope

Results Generation of LC/E Chimeras Containing the Localization Signalsfor LC/A

We have identified sequences in the N-terminus and C-terminus of theLC/A that are important for localization of the protein to the plasmamembrane. Deletion of the first 8 amino acids at the N-terminus causescomplete loss of plasma membrane localization, with the LC/A(ΔN8)localizing in the cytoplasm with nuclear exclusion. Disruption of thedi-leucine motif at the C-terminus produces also changes inlocalization. To validate those signals we generated chimeras betweenthe LC/A and the LC/E, since both cleave the same substrate at differentsites, but have different subcellular localization and duration ofaction. LC/E localizes to the cytoplasm and lasts 1-2 weeks. Wegenerated the following constructs using the native beluga LC/E sequenceof SEQ ID NO: 136: LC/E(N-LCA) of SEQ ID NO: 138, LC/E(ExxxII) of SEQ IDNO: 122, LC/E(ExxxLL) of SEQ ID NO: 140, LC/E(N-LCA/ExxxII) of SEQ IDNO: 123, and LC/E(N-LCA/ExxxLL) of SEQ ID NO: 142 (FIG. 3 and Table 7)in order to analyze the effect of each signal by itself, and the effectof both signals combined. These constructs were transfected into thehuman neuroblastoma cell line SH-SY5Y. Cell lysates were prepared andthe activity of the mutants was analyzed with the SMI-81 antibody thatrecognizes both cleaved and intact SNAP25 (FIG. 4).

TABLE 7 Name Contains GFP attached to: GFP-LCE SEQ ID NO: 136, aWild-type-LC/E GFP-LCE Nterm LCA SEQ ID NO: 138, a LC/E with the firsteight amino-acid of LC/A at the N-terminus GFP-LCE (ExxxII) SEQ ID NO:122, a LC/E with a di-isoleucine motif in C-terminal regiondi-isoleucine can substitute for leucine in the motif) GFP-LCE (ExxxLL)SEQ ID NO: 140, a LC/E with a di-leucine motif in C-terminal regionGFP-LCE Nterm LCA (ExxxII) SEQ ID NO: 123, a LC/E with the first eightamino-acid of LC/A at the N-terminus and di-isoleucine motif inC-terminal region GFP-LCE Nterm LCA SEQ ID NO: 142, a LC/E with thefirst eight amino-acid of LC/A at the (ExxxLL) N-terminus and dileucinemotif in C-terminal region

All the chimeras expressed in SH-SY5Y are able to cleave SNAP25₂₀₆ intoSNAP25₁₈₀, and they do not cleave the substrate at any other sites (FIG.4). The preliminary data from the two experiments performed showsdifferent levels of activity of the chimeras when compared to nativeLC/E. We could not distinguish if those differences are due to lower orhigher levels of expression of the mutants, or to true changes oncatalytic activity, because there was not enough material to run animmunoprecipitation to detect the chimeric LC/Es. FIG. 5 shows theresults of a new set of three independent experiments transfecting thechimeric LC/Es into PC12 and SH-SY5Y cells. The levels of expression ofeach construct are different but all of them retain catalytic activitytowards the cleavage of SNAP25. For some of the constructs the catalyticactivity seemed diminished when compared with the wild-type LC/E butthose changes can only be confirmed by expressing the proteinrecombinantly and performing ELISA or GFP assays.

Work previously published by Fernandez-Salas, Steward et al. (PNAS 101,3208-3213, 2004) showed the sub-cellular localization of GFP-LC/A andGFP-LC/E proteins in differentiated PC-12 cells (FIG. 6). GFP-LC/Alocalized in a punctate manner in specific areas in the plasma membraneof the cell body and the neurites, with no localization of GFP-LC/Aprotein in the cytoplasm of the cells (FIG. 6A.). GFP-LC/E showedcytoplasmic localization with nuclear exclusion. Cells displayed roundedmorphology and lack of neurites even in differentiation medium (FIG.6B.).

The results in FIG. 6 demonstrate that the light chains from BoNTserotypes A and E are directed to distinct sub-cellular compartments. Toidentify sequences of importance in directing LC/A localization to theplasma membrane, PC-12 and SH-SY5Y cells were transiently transfectedwith GFP-LC/E and GFP-LC/E (N-LCA/ExxxLL) plasmid constructs.Transfected SH-SY5Y cells were selected in growth medium containing 0.5mg/ml G418 for 2 days before staining with the Anti-SNAP25 and Anti-GFPantibodies. The transfected PC-12 cells were exposed to selection mediumfor 2 days followed by differentiation medium (containing NGF at 50ng/ml final concentration) for 24 hours before staining.

Staining for the GFP portion of the LC/E chimera demonstrated that mostof the GFP-LCE(N-LCA/ExxxLL) protein was localized at the plasmamembrane in a punctuate manner (FIGS. 7A and C), similar to thepreviously reported GFP-LC/A localization (FIG. 6A). The mousemonoclonal antibodies to SNAP25₁₈₀ used in this staining have highbackground staining but the cells expressing LC/E displayed a strongersignal. Moreover, cells expressing the GFP-LCE (N-LCA/ExxxLL) chimeracontain SNAP25₁₈₀ that remains in the cytoplasm.

GFP staining shown in FIGS. 8A and 8C demonstrates the punctatelocalization of GFP-LC/E (N-LCA/ExxxLL) protein at the plasma membraneof the cells, similar to the GFP-LC/A localization presented in FIG. 6A.The cells that are expressing the GFP-LC/E (N-LCA/ExxxLL) chimeraprotein (indicated by arrows corresponding to cells ‘a’, ‘b’ and ‘e’ inFIGS. 8A and 8C) do not show staining with Anti-SNAP25₂₀₆ antibody(cells ‘a’, ‘b’ and ‘e’ in FIGS. 8B and 8D). This confirms theproteolytic activity of the GFP-LC/E (N-LCA/ExxxLL) protein expressed inthese cells, as demonstrated by its ability to cleave SNAP25₂₀₆. On thecontrary, cells depicted ‘c’ and ‘d’ in FIG. 8C that are not expressingthe GFP-LC/E (N-LCA/ExxxLL) protein give a good signal for the presenceof full length SNAP25₂₀₆ protein as indicated by cells ‘c’ and ‘d’ inFIG. 8D.

The GFP-LC/E protein is shown to localize to a structure within thecytoplasm in a punctate manner as previously reported inFernandez-Salas, Steward et al. and Fernandez-Salas, Ho et al. (MovementDisorders 19, S23-S34., 2004) and shown in FIG. 6B. SNAP25₁₈₀ proteinalso localizes in the cytoplasm of cells expressing GFP-LCE and appearsto be in a granular structure (FIG. 9B).

SH-SY5Y Localization of GFP-LC/E

GFP-LC/E protein localizes in the cytoplasm of SH-SY5Y cells. Notice thelarge area of GFP exclusion in the nuclei of both cells shown in FIG.10.

The GFP-LCE (N-LCA/ExxxLL) chimera was also expressed in SH-SY5Y cells.Staining with an anti-GFP antibody (FIG. 11) demonstrated localizationat the plasma membrane, similar to that seen for the chimera expressedin PC-12 cells.

The plasmid encoding chimeric GFP-LC/E (N-LCA/ExxxLL) was transfectedinto the human neuroblastoma cell line SH-SY5Y. The chimeric protein wasexpressed and dishes were fixed for immunostaining. Cells stainedpositive for GFP (monoclonal antibody) (indicated by arrows in FIGS. 12Aand 12C) show the GFP-LCE (N-LCA/ExxxLL) protein localized to the plasmamembrane. Full length SNAP25₂₀₆ protein is not detected in cellsexpressing the GFP-LCE (N-LCA/ExxxLL) protein as indicated by the arrowsin FIGS. 12B and 12D. This suggests the GFP-LCE (N-LCA/ExxxLL) chimeraprotein is functional and cleaves SNAP25₂₀₆.

To further confirm activity the transfected cells were also stained withthe GFP and the monoclonal antibodies to SNAP25₁₈₀. Staining for GFP(polyclonal antibody) showed the GFP-LC/E (N-LCA/ExxxLL) proteinlocalized at the plasma membrane as shown in the previous figure (FIG.13A). The 1A3A7 ascites mouse monoclonal antibody used to stainSNAP25₁₈₀ has high background staining and is very weak (arrows in FIG.13B indicate groups of cells positive for SNAP25₁₈₀ staining. Thisantibody was used at 1:100 dilution in this early experiment. However,cells expressing the GFP-LCE (N-LCA/ExxxLL) protein in FIG. 13A containSNAP25₁₈₀ in the cytoplasm as indicated by arrows in FIG. 13B.

The GFP-LC/E fusion protein has previously been demonstrated to localizein the cytoplasm of PC12, HIT-T15, and HeLa cells. Adding the N-terminalLC/A signal (8 amino acids of SEQ ID NO: 120) and the C-terminaldi-leucine motif of LC/A (SEQ ID NO: 145) into the LCE protein sequence(GFP-LCE (N-LCA/ExxxLL) dramatically changed the sub-cellularlocalization of LC/E. Adding the LC/A localization signals to LC/Edirected localization of the LC/E (N-LCA/ExxxLL) chimera to the plasmamembrane, validating these motifs/sequences as important signals forLC/A localization. Cleavage of SNAP25 by LC/E was detected usingantibodies to the N-terminus of SNAP25. Co-staining with antibodies tofull length SNAP25₂₀₆ demonstrated a loss of intact SNAP25 in cellsexpressing the functional GFP-LCE (N-LCA/ExxxLL) chimera protein.SNAP25₁₈₀ protein was also detected in the cytoplasm of cells expressingGFP-LCE (N-LCA/ExxxLL), indicating LC/E SNAP25 proteolysis. Further workwill need to be done to optimize staining of SNAP25₁₈₀ cleavage productin the paraformaldehyde fixed cells because the anti-SNAP25₁₈₀antibodies used in this study were weak. To identify compartments wherethe LC/A, LC/E and LC/E (N-LCA/ExxxLL) proteins reside, a panel of dyesand antibodies specific for cytoplasmic organelles and plasma membraneproteins (channels and receptors) will be employed. PC-12 cells havebeen transfected with wild-type GFP-LC/E, GFP-LC/E (ExxxLL), GFP-LC/E(ExxxII), GFP-LC/E (N-LCA), GFP-LC/E (N-LCA/ExxxII) and wild-typeGFP-LC/A constructs. Localization of these chimera proteins will confirmsequences of importance in LC/A localization. The LC/E chimeracontaining the N-terminus of the LC/A and the di-leucine motif presentsa very distinct localization and may constitute a LC/E with a longerduration of action.

While this invention has been described with respect to various specificexamples and embodiments, it is to be understood that the invention isnot limited thereto and that it can be variously practiced with thescope of the following claims. All articles, references, publications,and patents set forth above are incorporated herein by reference intheir entireties.

1. A modified toxin comprising a modified light chain of a botulinumtoxin type E, wherein the modification to the botulinum toxin type Elight chain is the addition of one or more amino acid sequencescomprising SEQ ID NO: 120 within the N-terminal 30 amino acids of awild-type botulinum toxin type E light chain of SEQ ID NO: 136 and theaddition of one or more leucine-based motifs of SEQ ID NO: 111 withinthe C-terminal 50 amino acids of a wild-type botulinum toxin type Elight chain of SEQ ID NO: 136, wherein the addition of the amino acidsequence comprising SEQ ID NO: 120 increases biological half-life of themodified toxin relative to an identical modified toxin without theadditional amino acid sequence comprising SEQ ID NO: 120, and whereinthe addition of the leucine-based motif of SEQ ID NO: 111 increasesbiological half-life of the modified toxin relative to an identicalmodified toxin without the additional leucine-based motif comprising SEQID NO:
 111. 2. The modified toxin of claim 1, wherein the addition ofone or more amino acid sequences comprising SEQ ID NO: 120 is within thefirst 12 amino acids of the N-terminal of a wild-type botulinum toxintype E light chain of SEQ ID NO: 136, and the addition of one or moreleucine-based motifs of SEQ ID NO: 111 is within the last 27 amino acidsof the C-terminal of a wild-type botulinum toxin type E chain of SEQ IDNO:
 136. 3. The modified toxin of claim 1, wherein the addition of oneor more amino acid sequences comprising SEQ ID NO: 120 is within thefirst 8 amino acids of the N-terminal of a wild-type botulinum toxintype E light chain of SEQ ID NO: 136, and the addition of one or moreleucine-based motifs of SEQ ID NO: 111 is within the last 23 amino acidsof the C-terminal of a wild-type botulinum toxin type E light chain ofSEQ ID NO:
 136. 4. The modified toxin of claim 1, wherein the additionof one or more amino acid sequences comprising SEQ ID NO: 120 is withinthe first 8 amino acids of the N-terminal of a wild-type botulinum toxintype E light chain of SEQ ID NO: 136; and the addition of one or moreleucine-based motifs of SEQ ID NO: 111 is prior to the last 16 aminoacids at the C-terminal of a wild-type botulinum toxin type E lightchain of SEQ ID NO:
 136. 5. The modified toxin of claim 1, 2, 3, or 4,wherein the toxin further comprises a heavy chain of a Clostridialtoxin.
 6. The modified toxin of claim 5, wherein the heavy chain is aheavy chain of a botulinum toxin type A, botulinum toxin type B,botulinum toxin type C₁, botulinum toxin type D, botulinum toxin type E,botulinum toxin type F, or botulinum toxin type G.
 7. The modified toxinof claim 5, wherein the heavy chain is a heavy chain of a botulinumtoxin type A.
 8. The modified toxin of claim 5, wherein the heavy chainis a heavy chain of a botulinum toxin type E.
 9. The modified toxin ofclaim 1, wherein the leucine-based motif added is SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 10, or SEQ ID NO:
 12. 10. The modified toxin of claim1, wherein the modified botulinum toxin type E light chain is SEQ ID NO:126.
 11. The modified toxin of claim 1, wherein the modified botulinumtoxin type E light chain is SEQ ID NO:
 131. 12. A modified toxincomprising a modified light chain of SEQ ID NO: 136, wherein one or moreof SEQ ID NO: 120 occur within the N-terminal 30 amino acids of thelight chain, and one or more of SEQ ID NO: 111 occur within theC-terminal 50 amino acids of the light chain wherein the addition of oneor more SEQ ID NO: 120 increases biological half-life of the modifiedtoxin relative to an identical modified toxin without the addition ofone or more SEQ ID NO: 120, and wherein the addition of one or more SEQID NO: 111 increases biological half-life of the modified toxin relativeto an identical modified toxin without the addition of one or more SEQID NO: 111.